Better Labs and Prep Rooms

Better Labs and Prep Rooms kdorfman Thu, 06/04/2009 - 17:01

Here are the recipes for making solutions and doing stuff in the ISB.

2022 Summer

2022 Summer kdorfman Fri, 04/29/2022 - 16:17
  • Get Mary authenticated on Wahoo
  • Set up "permanent" groups for orders and cell lines spreadsheets
  • Empty growth chambers & take soil to greenhouse for compost
  • clean oculars on dissecting scopes in 364
  • Tie up cables 360
  • Tie up cables in 364
  • Tie up cables in 264
  • is -80 on generator power
  • ask Rolf about centrifuges and vortex mixers
  • Ladders / DNA Standards Inventory (1 Kb for QBoC)
  • Defrost freezer 266A
  • Defrost freezer 362A
  • Clean microscopes 264 * Clean microscopes 364
  • Sharpen forceps
  • Refill Vaseline syringes
  • organize mini-drawers
  • Pipette rodeo

  • Pour plates

    • 100 LB
    • 250 60 YED
    • 150 MV
    • 70 MVADE
    • 60 YeKAc

  • Inventory and replenish

    • order Aci1, Mlu1, XBa1, 1 Kb ladder from NEB #B7025
    • major equipment (with purchasing)
    • mini-preps
    • tips
    • tubes
    • sterile toothpicks
    • sterile glass beads
    • slides & coverslips
  • autoclave coverslips
    • cell culture reagents
    • genetics reagents & equipment
    • 161H materials
  • Prep tissue culture room
  • Move genetics from 364 to 264
  • Schedule fluorescence scope repair
  • Clear out 366A for Animal Science
  • Move -80 from Lederle to ISB
  • Move into new -80
  • Inventory E. coli
  • Move LN2 to371

Chemical delivery address

Chemical delivery address kdorfman Tue, 01/07/2020 - 21:47

University of Massachusetts

Room 179 Lederle Graduate Research Tower

710 North Pleasant Street

Amherst, MA 01003-9305

ATTN: Katherine Dorfman, ISB 362A

Contact Glenda Pons at EH&S regarding deliveries
gpons@ehs.umass.edu (413) 577-3631

Computer admin

Computer admin kdorfman Fri, 07/29/2022 - 18:39

username: .\admin

pw :

To find the computer's name:
rt click start menu
run
cmd
ipconfig /all

Courses

Courses kdorfman Thu, 01/11/2024 - 18:27

Courses taught in ISB

Bio 499CB (Loomis Cancer)

Bio 499CB (Loomis Cancer) kdorfman Thu, 09/08/2022 - 20:59

Kari Loomis honors thesis seminar

Librarian does workshop on library research.

We do Bradford assay lab:

Explain Bradford assay.

Add some Bradford reagent to some BSA - Ask how much protein?

Need standards to compare to!

  • Bradford concentrate from Bio-Rad 500-0006
  • dilute 1:4 with water
  • Make BSA solution 0.1 mg/mL (dilute from the test 2 m/mL test solution
  • Students share a plate:
    • one uses top row
    • one uses bottom row
  • write a program on PS2

Student protocol:

  • Make a 2-fold dilution series of the BSA
    • 0.5 mL water in each tube (except the first)
    • 1 mL BSA into first tube
    • .5 mL to next tube, mix, transfer to next, etc.
  • Put 160 uL BSA into each well
  • put 160 uL water in one well. (Why?)
  • Add 40 uL Bradford
  • Read plate

Data management

  • Stupid Excel Tricks
  • Art of the standard curve
  • Make your own std curve.
  • How would you estimate the concentration of protein in a solution that had an Abs of:
    • 0.5
    • 0.4
    • 3
Raw data
Bradford processed data
preya_tina_9-9_ps2.xls
Nabil Amira Bradford_ps2.xls
pipette good_ps2.xls
Pipette Champions_ps2.xls
abe_cal_BRADplate_ps2.xls

Marine Biology

Marine Biology kdorfman Mon, 01/15/2024 - 17:32

Bio 424 Spring 2024 (Akiko Okusu)

Materials Request

Prep Instructions

Sea Water for 424

Sea Water for 424 kdorfman Mon, 01/15/2024 - 18:14

2 carboys SeaWater (31-35ppt salinity):

  • 31-35g InstantOcean Reef Salt in 1 L
  • Submerge a bubbler or a pump so that it is constantly being mixed

F2 Medium for algae (424)

F2 Medium for algae (424) kdorfman Mon, 01/15/2024 - 18:16

5 liters of f/2 medium for algae.

Re-inoculate once during week of 1/15

TA & Students will re-inoculate once monthly

50 mL/month x 4 months x 16 students = 3200 mL

Make 4 L for the semester.

To make (in a sterile hood)L F/2 medium for algae
add: mL NaNO3 (75g/L)
add: mL NaHPO4 (5 g/L)
add: mL trace minerals
add: mL vitamins
add: mL kanamycin
add: mL ampicillin
add: mL streptomycin

Filter sterilize

F/2 medium for algae

Perfect Plant 2024

Perfect Plant 2024 kdorfman Wed, 01/10/2024 - 21:51

Bio 427 - Madelaine Bartlett

Wednesdays

Date subject prep
2/7 plant architecture Find the leaf. lycophytes, bryophytes, diversity, alstoemeria, Ruscus
2/14 ferns and lycophytes ???? clv1 and wus mutants? from maize, tomato, arabidopsis; moss phyllotaxy
2/21 seed plants life cycles - moss gametophytes, sporophytes; tree thinking/traits on a tree. moss gametophytes, fern gametophytes, prepared slides
2/28 individual plant 1
3/6 fruit diversity fruit diversity (grocery store) including Citrus diversity; fas PCR, fruit description
3/13 exam 1
3/20 spring break
3/27 ABC model arabidopsis and maize ABC(E) mutants
4/3 individual plant 2
4/10 floral diversity grocery store floral diversity
4/17 exam 2
4/24 leaf development patterning mutants - ask Aman, Annis
5/1 leaf diversity Leaf morphological and anatomical diversity (C3 and C4), practice making hand sections
5/8 presentations

Prep pages

2024/02/07

2024/02/07 kdorfman Thu, 01/11/2024 - 18:47

Perfect Plant Lab 1

Topic: Major lineages of land plants, basics of plant architecture

Plants needed: NHC diversity; maize, arabidopsis, tomato

Prepared slides: Meristems

Materials:

  • dissection tools
  • hand section materials
  • microscopes

2024/02/14

2024/02/14 kdorfman Thu, 01/11/2024 - 19:01

Perfect Plant Lab 2

Topic: Fern lifecycle

Resources: C-fern Manual

Materials: dissection and hand section materials, microscopes

Plants: C-fern gametophytes; NHC diversity

Prep: Sow Arabidopsis ABC mutants

2024/02/21

2024/02/21 kdorfman Thu, 01/11/2024 - 19:06

Perfect Plant Lab 3

Topic: Seed plants

Plants needed: meristem mutants; NHC diversity

Prepared Slides: gametophytes; embryos

Materials: dissection and hand section materials, microscopes; maybe stuff for pollen tube growth

2024/02/28

2024/02/28 kdorfman Tue, 01/16/2024 - 21:37

Individual Plant Day 1

2024/03/27

2024/03/27 kdorfman Sun, 01/28/2024 - 15:54

Arabidopsis mutants:

Stock # tape color allele gene
CS3085 teal ap3-1 AT3G54340
CS3082 green ap2-1;ap2-2 AT4G36920
CS8066 red (clv3-2) AT2G27250
CS45 blue clv1-1 AT1G75820
CS77 lavender pi-1 AT5G20240
CS25 pink ag-1 AT4G18960
CS6291 orange ap1-7 AT1G69120
CS15 yellow wus-1 AT2G17950

Display Easels

Display Easels kdorfman Mon, 05/12/2014 - 15:17

Looking for a better way to display student posters, especially at the luncheon for graduating seniors.

minimalist 1 piece easel $136 black 4'7" ($1468/12)

minimalist bi-fold display easels 6' black bamboo $50

how-to make bi-fold display easel

Skyscraper Mightee Mount $168/pair

link title

Electronics and Equipment

Electronics and Equipment kdorfman Wed, 10/19/2011 - 16:27
isotemp_calibration.xlsx
isotemp_plug.jpeg

Computers, servers, etc.

Computers, servers, etc. kdorfman Wed, 08/19/2009 - 16:55

The OIT wireless network is installed throughout the ISB. Make sure you have turned on AirPort or your wireless receiver, and sign in using your OIT account username and password.

Set up your bcrc account here: https://wahoo.nsm.umass.edu/passwd/

Computers that are plugged into the network in the biology labs are on the BCRC server; sign in as yourself (after you have set up your account).

Getting to Wahoo files from home

Getting to Wahoo files from home kdorfman Sat, 10/29/2011 - 17:15
  • Get and install Filezilla (see here).
  • Open FileZilla
  • Open Site Manager from the File Menu:
    • Host name: wahoo.nsm.umass.edu
    • Username: your bcrc username
    • password: your bcrc password
    • port: 22
    • Protocol: SFTP
    • Logon Type: ask for passwork
    • User: your BCRC username
  • Click Connect
  • Enter your BCRC username and password
  • The first time, you get a warning about an unknown host. Check the box next to Always trust this host, add this key to the cache, and click OK
  • You should be in a directory called something like /u1/home/bio/username (with your bcrc username, of course).

  • Clear the Remote site and type the appropriate one of these:

    • /export/quantbiol
    • /export/Bioimaging
    • /export/mboms

  • Enlarge the absurdly small window under the remote site bar and scroll to find your microscope folder. Move files between local and remote folders by drag-and-drop, or by right click and upload (local to remote) or download (remote to local)

Voilà!

Printing

Printing margaret Wed, 10/12/2011 - 15:35

Go to the Biology, BCRC web site. Hit drop down menu for Undergraduate, print release ISB.

Resources-print release.

Sign in, select the printer, release, print.

Printers are Xerox ColorQube 8570

264 Nifiloli

360 Nupani

364 Nukapu

368 Ngawa

Inks (2 packs) from Gov Connection $138.37

yellow 108R00928

cyan 108R00926

magenta 108R00927

black 4 pack 108R00930

Xerox 108R00966 Rainbow pack (1 each CYMK) from Spare Parts Warehouse @$74.95

Printing in Biology ISB space 2019.pdf

Screen Capture

Screen Capture rootlet Fri, 04/02/2010 - 14:46

You can capture still images of videos of anything on the computer screens that Biology supports.

Capture Stills

To capture still images, you can use some magic keystrokes:

Capture whole screen: Command + Shift + 3

Capture region: Command + Shift + 4 to turn cursor to cross-hairs, then select region.

There is also an application in the Utilities folder called "Grab" that will let you set some options or get images just of particular windows.

Capture Video

To capture video, you need to use the command-line. Videos of the entire screen are very large. You might want to use the System Preferences to set the screen resolution lower before starting to capture video. We're using the vnc2flv project. Start up Terminal (Also in the Utilities folder, but there a shortcut in the Dock). You can use two commands to start recording video. With any luck, it will be as simple as this:

First, go to the Desktop to save your work there: delfeno:~ sbrewer$ cd Desktop

Then start x11vnc: delfeno:Desktop sbrewer$ x11vnc &

You'll see a bunch of output -- once the output stops, hit return to get the prompt back, then type: delfeno:Desktop sbrewer$ flvrec.py

It will record video and save it in a file on the Desktop until you tell it to stop. You tell it to stop by typing Control + C. You should end up with a file on the Desktop called something like "out201004020900.flv". You can open this file using VLC to watch the video. To import the file into iMovie, you need to transcode the file into something iMovie understands: delfeno:Desktop sbrewer$ ffmpeg -i out201004020900.flv -sameq out201004020900.mov

The .mov file can be imported into iMovie, where you can edit the clip, add a sound-track, or combine with other videos.

Below is a complete session so you can see all the parts put together.

   Last login: Fri Apr  2 09:21:58 on ttys006
   delfeno:~ sbrewer$ cd Desktop
   delfeno:Desktop sbrewer$ x11vnc &
   [1] 37779
   delfeno:Desktop sbrewer$ 02/04/2010 09:44:17 MacOS X: set -connect file to /tmp/x11vnc-macosx-remote.sbrewer
   ###############################################################
   #@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@#
   #@                                                           @#
   #@  **  WARNING  **  WARNING  **  WARNING  **  WARNING  **   @#
   #@                                                           @#
   #@        YOU ARE RUNNING X11VNC WITHOUT A PASSWORD!!        @#
   #@                                                           @#
   #@  This means anyone with network access to this computer   @#
   #@  may be able to view and control your desktop.            @#
   #@                                                           @#
   #@ >>> If you did not mean to do this Press CTRL-C now!! <<< @#
   #@                                                           @#
   #@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@#
   #@                                                           @#
   #@  You can create an x11vnc password file by running:       @#
   #@                                                           @#
   #@       x11vnc -storepasswd password /path/to/passfile      @#
   #@  or   x11vnc -storepasswd /path/to/passfile               @#
   #@  or   x11vnc -storepasswd                                 @#
   #@                                                           @#
   #@  (the last one will use ~/.vnc/passwd)                    @#
   #@                                                           @#
   #@  and then starting x11vnc via:                            @#
   #@                                                           @#
   #@      x11vnc -rfbauth /path/to/passfile                    @#
   #@                                                           @#
   #@  an existing ~/.vnc/passwd file from another VNC          @#
   #@  application will work fine too.                          @#
   #@                                                           @#
   #@  You can also use the -passwdfile or -passwd options.     @#
   #@  (note -passwd is unsafe if local users are not trusted)  @#
   #@                                                           @#
   #@  Make sure any -rfbauth and -passwdfile password files    @#
   #@  cannot be read by untrusted users.                       @#
   #@                                                           @#
   #@  Use x11vnc -usepw to automatically use your              @#
   #@  ~/.vnc/passwd or ~/.vnc/passwdfile password files.       @#
   #@  (and prompt you to create ~/.vnc/passwd if neither       @#
   #@  file exists.)  Under -usepw, x11vnc will exit if it      @#
   #@  cannot find a password to use.                           @#
   #@                                                           @#
   #@                                                           @#
   #@  Even with a password, the subsequent VNC traffic is      @#
   #@  sent in the clear.  Consider tunnelling via ssh(1):      @#
   #@                                                           @#
   #@    <a href="http://www.karlrunge.com/x11vnc/#tunnelling">http://www.karlrunge.com/x11vnc/#tunnelling</a>            @#
   #@                                                           @#
   #@  Or using the x11vnc SSL options: -ssl and -stunnel       @#
   #@                                                           @#
   #@  Please Read the documention for more info about          @#
   #@  passwords, security, and encryption.                     @#
   #@                                                           @#
   #@    <a href="http://www.karlrunge.com/x11vnc/faq.html#faq-passwd">http://www.karlrunge.com/x11vnc/faq.html#faq-passwd</a>    @#
   #@                                                           @#
   #@  To disable this warning use the -nopw option, or put     @#
   #@  the setting in your ~/.x11vncrc file.                    @#
   #@                                                           @#
   #@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@#
   ###############################################################
   02/04/2010 09:44:19 x11vnc version: 0.9.9 lastmod: 2009-12-21  pid: 37779
   02/04/2010 09:44:19 XOpenDisplay(":0.0") failed.
   02/04/2010 09:44:19 Trying again with XAUTHLOCALHOSTNAME=localhost ...
   02/04/2010 09:44:19 Continuing without X display in -rawfb mode.
   02/04/2010 09:44:19 macosxCG_init: initializing display.
   02/04/2010 09:44:19 console_guess: file is /dev/null
   02/04/2010 09:44:19 console_guess returned: map:macosx:/dev/null@1680x1050x32:ff0000/ff00/ff
   02/04/2010 09:44:19 raw fb is non-regular file: /dev/null
   02/04/2010 09:44:19 rawfb: macosx fb: /dev/null
   02/04/2010 09:44:19    w: 1680 h: 1050 b: 32 addr: 0x1860000 sz: 7056000
   02/04/2010 09:44:20 initialize_screen: fb_depth/fb_bpp/fb_Bpl 24/32/6720
   02/04/2010 09:44:20 
   02/04/2010 09:44:20 Raw fb at addr 0x1860000 is 32bpp depth=24 true color
   02/04/2010 09:44:20 
   02/04/2010 09:44:20 Autoprobing TCP port 
   02/04/2010 09:44:20 Autoprobing selected port 5900
   02/04/2010 09:44:20 fb read rate: 10 MB/sec
   02/04/2010 09:44:20 Manually set num_buttons to: 5
   02/04/2010 09:44:20 screen setup finished.
   02/04/2010 09:44:20 
   02/04/2010 09:44:20 WARNING: You are running x11vnc WITHOUT a password.  See
   02/04/2010 09:44:20 WARNING: the warning message printed above for more info.
   02/04/2010 09:44:20 
   
   The VNC desktop is:      delfeno.bio.mor.nsm:0
   PORT=5900
   
   delfeno:Desktop sbrewer$ flvrec.py 
   start recording
   02/04/2010 09:44:26 Got connection from client 127.0.0.1
   02/04/2010 09:44:26   other clients:
   02/04/2010 09:44:26 macosxCG_callback: register
   02/04/2010 09:44:26 incr accepted_client=1 for 127.0.0.1:53061  sock=5
   02/04/2010 09:44:26 Client Protocol Version 3.8
   02/04/2010 09:44:26 Protocol version sent 3.8, using 3.8
   02/04/2010 09:44:26 rfbProcessClientSecurityType: executing handler for type 1
   02/04/2010 09:44:26 rfbProcessClientSecurityType: returning securityResult for client rfb version >= 3.8
   02/04/2010 09:44:26 Pixel format for client 127.0.0.1:
   02/04/2010 09:44:26   32 bpp, depth 8, big endian
   02/04/2010 09:44:26   true colour: max r 255 g 255 b 255, shift r 24 g 16 b 8
   02/04/2010 09:44:26 Using raw encoding for client 127.0.0.1
   02/04/2010 09:44:26 copy_tiles: allocating first_line at size 54
   02/04/2010 09:44:27 client_set_net: 127.0.0.1  0.0007
   ^Cstop recording
   delfeno:Desktop sbrewer$ 02/04/2010 09:44:33 client_count: 0
   02/04/2010 09:44:33 viewer exited.
   02/04/2010 09:44:33 deleted 53 tile_row polling images.
   02/04/2010 09:44:33 macosxCG_callback: unregister
   
   [1]+  Done                    x11vnc
   delfeno:Desktop sbrewer$ ffmpeg -i out201004020944.flv -sameq out201004020944.mov
   FFmpeg version 0.5.1, Copyright (c) 2000-2009 Fabrice Bellard, et al.
     configuration: --enable-libmp3lame --enable-libfaac --enable-nonfree
     libavutil     49.15. 0 / 49.15. 0
     libavcodec    52.20. 1 / 52.20. 1
     libavformat   52.31. 0 / 52.31. 0
     libavdevice   52. 1. 0 / 52. 1. 0
     built on Mar 31 2010 12:11:13, gcc: 4.0.1 (Apple Inc. build 5493)
   
   Seems stream 0 codec frame rate differs from container frame rate: 1000.00 (1000/1) -> 12.00 (12/1)
   Input #0, flv, from 'out201004020944.flv':
     Duration: 00:00:07.16, start: 0.000000, bitrate: N/A
       Stream #0.0: Video: flashsv, bgr24, 1696x1056, 12 tbr, 1k tbn, 1k tbc
   Output #0, mov, to 'out201004020944.mov':
       Stream #0.0: Video: mpeg4, yuv420p, 1696x1056, q=2-31, 200 kb/s, 90k tbn, 12 tbc
   Stream mapping:
     Stream #0.0 -> #0.0
   Press [q] to stop encoding
   frame=   87 fps= 42 q=0.0 Lsize=    7277kB time=7.25 bitrate=8222.8kbits/s    
   video:7276kB audio:0kB global headers:0kB muxing overhead 0.019529%
   delfeno:Desktop sbrewer$

Updating the computers-radmind

Updating the computers-radmind margaret Wed, 10/12/2011 - 15:41

Using the apple drop down, logout.

When the user name and password comes up, type in: radmind, enter, enter.

The computer will update.

Important! Connect the laptops to an etherternet cable to run radmind.
Do not run from wireless connection!

Major equipment

Major equipment kdorfman Wed, 07/15/2009 - 20:57

Autoclaves in 261, 361

Dishwashers in 261, 361 (Belimed WD 230 SN 998360086004)

Freezers (-20) in 366A, 262A

Freezer (-80) in 262A

Percival Incubators in 364 (originally from the Markstein lab) I36NL SN17922. 04. 12I

Plant tissue culture incubator in 373

2 Arabidopsis growth chambers in 373

Convection Oven (cord broken summer 2022)

Leica cryostat CM1859 S/N 5814

  • in 264 (was used in histology and as emergency cryostat for various research labs)
  • maybe moving to Morrill equipment room

Minus 80 (from Biochem storage) SANYO VIP -86 (SN #41115285) in 364

Belimed WD 230 manual
Sanyo -80 manual
Percival Intellus manual

Convection Oven

Convection Oven margaret Fri, 10/14/2011 - 16:01

Motic Camera & Software

Motic Camera & Software margaret Wed, 10/12/2011 - 16:03

Plug your camera into a USB port on the back of the computer

Open Finder

Go down to applications

Select Motic Images Plus

Motic Images Plus

With camera attached you will see an image.

If not try:

     File
     New
     Live Video
using_the_motic_camera.docx
motic_cam_in_situ.jpg
putting_adapter_on_motic_cam.jpg
putting_motic_on_microscope.jpg

Projectors

Projectors kdorfman Wed, 06/17/2009 - 14:50

There are two different kinds of projectors in the ISB biology/biochemistry lab wing:

Rooms 360 and 364 have the higher quality projectors.

In 360, the projector is turned on via the Crestron. Hold down the video button until the projector turns on, then press the pc button to switch to your computer.

The instructor's computer, at the central microscope, has a mini-DVI to DVI adaptor connection to the cable running up the central pole.

For best resolution from the instructor's computer, log in as bcrc, then go to system preferences>displays, and pick 1344 x 1008 on the projector. Switch to the monitor, and go to arrangement, set it to mirror images. Set the resolution to 1344 x 840.

Use the remote control (in the drawer at the window side of the room labeled "remote control") to turn the projector on and off if the Crestron doesn't seem to work.

To connect to the projector via VGA, use the connector on the Crestron.

To turn the projector off, press the video button until the light blinks.

In 364, there are two connections to the projector: under the window to the right of the screen (#2), and at the desk behind the projector (#1). Connecting to the jack at the window overrides the signal from the jack at the desk. The front jack should also give you a better image.

In room 264, there are two projector jacks: under the window to the right of the screen (#2), and at the desk behind the projector (#1). The jack at the front gives you better video quality, but if you need sound, you need to use the jack by the desk.

To reset the projector if is claiming input from video instead of computer, use the remote (in a labeled drawer near the jack at the front of the room). Or unplug the projector (the white cable from the projector to the ceiling outlet), wait a minute, then replug it.

Dissecting Scopes

Dissecting Scopes kdorfman Fri, 12/21/2018 - 19:34
Room base head how many
264 flat 445 1
mirror 445 4
360 flat 445 12
mirror trinoc (63X) 1
flat no head 6
364 flat no head 1
flat 445 11
mirror 445 3
368 mirror 445 6 (VASCI = 5)
mirror 645 17
mirror 745 2

Optima Plate Reader

Optima Plate Reader kdorfman Tue, 01/17/2017 - 19:12

Link to Programs here

sign in to the "student" account
user = .\student pw = student@ISB364

Find Optima Control in the start menu

Folders icon pinned to taskbar

Data files go to: This PC > Local Disc (C:) > users > student > documents
(also to backups)

Find protocols to import in: This PC > Local Disc (C:) > users > Public > Public Downloads > PS1_Protocols

Plate IDs (in the run menu)
ID1 is file name. Be sure to give a file name

To get the meta data on the output csv file:
Setup
Program configuration
preferences
define format

Set Preferences before running a script! See p 28 of user manual ii, below.

Setup > Program Configuration > Define Format > Filename and Path

File info

Overwrite, append, make new file with same name + number

Header:

  • no header - just the data - useful for files that have to be compiled
  • long header includes wavelength - just the right amount of info (USE THIS)
  • full header (Ex, Em wavelength, date, time, etc) way too much info
  • short header does not include wavelength
  • Danish headers writes the file name next to the row ID. (!?!)

Style

  • Table: no well numbers, or wavelength indicators, etc. Just the results in plate layout form.
  • Table with well numbers: puts the well IDs next to the reading (Very hard to read)
  • Table with well numbers (only measured wells)

  • Table with well numbers in plate layout style: USE THIS

  • List (Literally, a list. no plate info. all values in one column)

  • List with well numbers

    A B
    1 A01 10
    2 A02 12
    3 A03 11
    4 A04 13

  • List with well numbers (only measured wells): puts a - for a skipped well)

  • List sorted by wells (all cycles in one row, chromatics in separate blocks)
  • List sorted by wells with well numbers
  • List sorted by well numbers (only measured wells)
  • List sorted by wells (all cycles/intervals/channels/chromatics in one row)
  • List sorted by wells 2 " " " (only measured wells)

Script mode

For i = 1 to 72 This is the counter
id 1 = "BOD1" BOD1 is the file name
id 1 = "BOD1" i makes a separate file for each run, called BOD1_1. BOD1_2 etc.

Excitation filters Emission filters
340 520
485 570
492 590
530 620
544
584
595
optima.docx
0413b0001i_operating_manual_fluostar_polarstar_lumistar_optima.pdf
0413f0010a_software_manual_optima_part_i.pdf
0413f0011a_software_manual_optima_part_ii.pdf
0413f0016a_software_manual_optima_part_iiia.pdf
0413f0013a_software_manual_optima_part_iiib.pdf
0413f0014a_software_manual_optima_part_iv.pdf
0413f0015b_quick_guide_optima.pdf
bmg_filter_overview_scan_7a.xls

Plate Reader Programs

Plate Reader Programs kdorfman Thu, 10/13/2022 - 15:20

Look for Programs here

Desktop/This PC/Local Disc (C:)/Users/Public/Public Downloads/PS1_Protocols

or

On Pstar2, even though the files say Polar star 1):

Desktop/This PC/Local Disc (C:)/Users/Public/Public Downloads/polarstar1/PS1_Protocols

Program Layout method ex em vol
BUG OD 1 A&B Abs 595 . 250
BUG_FLUOR 2 A:H FL 486 520 .
BUG_OD_SHAKE 3 A:H ABS 595 . 200
FL-ABS-BY-2 4 C1:2, D ABS 485 . 150
REPEATABILITY 5 A & B ABS 485 . 150
SERIAL2&5_ABS 6 E1-5, F:H ABS 485 . 150
SERIAL2&5_FL 7 E1-5, F:H FL 485 520 .
REPEATAILITY_FL 8 A:B FL 485 520 .

Script for overnight growth and fluorescence

  1. Lab 3.1 (2022) counting bacteria: std curve OD vs population density ↩︎

  2. Lab 4.2 (2022) lac operon shake cells, then read OD for overnight growth curves – part of script “Grow&Glow” ↩︎

  3. Lab 4.2 (2022) lac operon after BugOD Shake,read FL for overnight growth curves– part of script “Grow&Glow” ↩︎

  4. Lab 1.2 serial dilution & plate reader sensitivity ↩︎

  5. Lab 1.2 pipetting consistency (ABS) ↩︎

  6. Lab 1.3 serial dilution; absorbance of fluorescein; std curve ↩︎

  7. Lab 1.3 serial dilution; fluorescence of fluorescein; std curve ↩︎

  8. (Lab 1.2) pipetting consistency (FL) not used in 2022 ↩︎

Script for lac operon lab

Script for lac operon lab kdorfman Mon, 10/24/2022 - 15:18

Script for overnight growth and fluorescence

Set Preferences inside the user dialog box before running a script!

Location: C:\Users\Public\Downloads\PS1_Protocols\E_Coli_Grow&Glow.btc

(or PS2)

Multiple measurements for 17.5 hours

;absorbance and fluorescence

st1:="BUG_OD_SHAKE"

st2:="BUG_FLUOR"

for i:=1 to 70 do begin

ID1:="BOD1"

ID2:= <protocol>

ID3:=<method>

R_Run "<st1>"

ID1:="BFL1"

ID2:= <protocol>

ID3:= <method>

R_Run "<st2>"

wait for 13 m

end;

beep

(Names for PS2 = BOD2, BFL2)

Other digital cameras

Other digital cameras kdorfman Mon, 01/15/2018 - 20:28

OptixCam

(uses toupview software)

Summit SK2-10X - 10.0MP - Digital USB 2.0 Microscope Camera - PC/MAC Compatible - Image Capture Software - Measuring Software (PC only) - C-Mount - 23mm Eyepiece Adapter

AmScope

10MP Windows & Mac OS Compatible Microscope Camera + Calibration Kit SKU: MA1000-CK

(download software from amscope)

http://www.amscope.com/software-download#tucs1

Equipment Lending

Equipment Lending kdorfman Mon, 06/19/2017 - 19:27
date borrowed by how many what for returned
6/19/17 Kit Kolbert 7 dissecting scopes flat bottom summer research intensive Markstein Lab 8/10/17
6/19/17 Kit Kolbert 12 fly tubing, pads, etc, + CO2 regulator summer reasearch intensive Markstein Lab 8/10/17
6/30/17 Rolf 1 old computer, spot camera, keyboard cannibalize for parts
6/25/18 Kit Kolbert 14 dissecting scopes flat bottom summer research intensive Markstein Lab 8/21/18
6/25/18 Kit Kolbert 14 fly tubing, pads, etc, + CO2 regulator summer reasearch intensive Markstein Lab 8/21/18
7/10/18 Kit Kolbert 7 forceps (turquoise) Summer research intensive Markstein Lab 8/21/18
7/10/18 Kit Kolbert 2 CO2 tank boots Summer research intensive Markstein Lab 8/21/18
7/10/18 Kit Kolbert 2 CO2 tank clamps & belts Summer research intensive Markstein Lab 8/21/18
7/10/18 Kit Kolbert 3 blue flashlights Summer research intensive Markstein Lab 8/21/18
7/10/18 Kit Kolbert 1 yellow filter Summer research intensive Markstein Lab 8/21/18
7/28/20 Xiang Li 1 microtome amd slide warmer from 264 Caicedo lab
8/31/20 Karen Dunphy 1 pipetmen B11 (1000x, 200x 20x) microbio lab
8/22/22 Akiko 0 blue flashlights and filters
8/10/22 Quentin (Rolf lab) 2 blue light box and camera adapter returned
9/15/22 Akiko Okusu 12 OptixCam class in Morrill fridays 9-12 (Craig needs them in ISB on 9/27&28, 10/4&5, 10/11&12 - mornings)) 11/25/22

HHMI Lab Manuals

HHMI Lab Manuals kdorfman Thu, 01/09/2014 - 19:38
bio190h-manual-2013-rwkd.pdf.pdf
dorfman-bio477h-2013-manual.pdf
2014-biobootcamp-dna-protocol.pdf
2014-biobootcamp-manual.pdf
dorfman-bio477h-2014.pdf
how_to_make_a_good_slide.pptx

Handouts

Handouts kdorfman Thu, 05/02/2019 - 18:51

CoolLED handout to sit next to microscope

cell culture ppt

pipet spreadsheet

Using transmitted light on the fluorescence scopes in 360

Using emitted light on the fluorescence scopes in 360

Aligning the transmitted light on the fluorescence scopes in 360

Adding a scale bar

Doing it all on the fluorescence scopes in 360

Chi Square Test for Counted Data
Colorometric Pipetting Exercise
Dilutions
Mean, standard deviation, and the t-test
Metric Prefixes
PowerPoint Presentations
Printing in Biology ISB space 2019_0.pdf
pipet buffer table.docx
Q-Test for the Outlying Data Point
2 mm stage micrometer
Cell Counting Using Hemocytometer Handout
CoolLED handout
Cell number estimates table

ISB Rooms

ISB Rooms kdorfman Thu, 01/19/2012 - 22:37

ISB hours

Email requests for 241H to pwhite@umass.edu

 

Classrooms

Classrooms kdorfman Thu, 01/19/2012 - 22:38

Conference rooms

Conference rooms kdorfman Thu, 01/19/2012 - 22:38

Mail room: 147

scheduling

Keys

Keys kdorfman Wed, 09/04/2019 - 15:04

IC-0 (red)

  • 241H (conference room)
  • 255 (chem labs)
  • 263A, 263B, 263C (chem office rooms)
  • 329 (classroom)
  • 361 (dishwashing)
  • 363A (animal sci classroom)
  • 363B, 363C (chem rooms)
  • 399R (3rd floor staff nook)

IC-1 (blue)

  • 261 (dishwashing)
  • 262A (biochem prep)
  • 264, 266A, 360, 362A, 364, 366A, 368 (bio lab doors )

IC-2

  • 147 (mail room)
  • 241 A-G (vestibule & offices)
  • 341 (chemistry office vestibule)

IC-3-A1X

  • 072 (basement storage room)

PK-2-22 (swipe access doors)

  • 266, 362, 366 (vestibules)
  • 369 (imaging)
  • 371 (tissue culture)

Lab Wing

Lab Wing kdorfman Thu, 01/19/2012 - 22:37

255 organic chem

260 biochem lab

261 glass wash & autoclave

262A biochem prep room (-80 freezer)

263 ice

264 biology flexible bench lab

266A biology prep room

268 biochem lab

275 ice

351 electrical breaker room

355 advanced chem labs

360 fluorescence microscopy (biology)

361 glass wash & autoclave

362A biology prep

363 ice

364 biology flexible bench lab

366A biology prep room (LN2)

368 molecular biology and sterile hoods (biology)

369 biology imaging

371 tissue culture (biology)

373 plant growth chambers and incubator

Lab Schedules

Lab Schedules kdorfman Fri, 11/18/2011 - 19:40

scheduling calendar

meeting_room_booking_system_copy.pdf

2020 Spring

2020 Spring kdorfman Tue, 12/20/2011 - 20:33
Dept # prof TA Room Day Time
Psych 430 Forger 264 M, F 2:30 - 4:25
NSB 618 K Cave, J Meyers 264 Th 11:00 - 1:00
Bio 523 E Connor B Olson 264 Tu, W 1:25 - 4:25
Bio 383H S Hazen T Friedrich (364)/368 M, W 12:30 - 4:30
Bio 284 Barlow A Ye 364/(368) Tu 1:15 - 4:25
Bio 197FH Riley, Patek P A Green 364 Tu, Th 9:00 - 12:00
Bio 499F Wadsworth Balchand 360, 371 Tu, Th 1:25 - 4:25
Bio 577 J Ross 360 M, W 1:25 - 3:25
Room Mon Tues Wed Thurs Fri
360 AM
PM Bio 577 1:25 - 3:25 Bio 499F 1:25 - 4:25 Bio 5771:25 - 3:25 Bio 499F 1:25 - 4:25
364 AM Bio 197H 9:00 - 12:00 Bio 197H 9:00 - 12:00
PM
368 AM
PM Bio 383H 12:30 - 4:30 Bio 284 1:15 - 4:25 Bio 383H 1:25 - 4:25
264 AM NSB 618 11:00 - 1:00
PM Psych 430 2:30 - 4:30 Bio 523 1:25 - 4:25 Bio 523 1:25 - 4:25 Psych 430 2:30 - 4:30

2011 Fall

2011 Fall kdorfman Tue, 12/20/2011 - 19:52
Class Room Day Time
Bioimaging 360 M, W 1:25 - 4:25
QBoC 364 Tu, Th 12:30 - 3:30
VASCI 290F 368 M 1:00 - 3:45
CM&BL 368 Th 12:30 - 4:30
CM&BL 360 F 12:30 - 2:30
Histology 264 Tu, W 1:25 - 4:25
Research Methods 360, 371 F 3:00 - 4:00
Room AM/PM Mon Tues Wed Thurs Fri
360 AM
PM Bioimage 1:25 - 4:25 Bioimage 1:25 - 4:25 CM&BL 12:20 - 2:30, Res Meth 3:00 - 4:00
364 AM
PM QBoC 12:20 - 3:30 QBoC 12:20 - 3:30
368 AM
PM VASCI 290F 1:00 - 3:30 CM&BL 12:20 - 4:30

2012 Fall

2012 Fall kdorfman Thu, 08/02/2012 - 19:30
Class Room Day Time
Bioimaging 360 M, W 1:25 - 4:25
Model Syst 360, 368 Tu, Th 1:25 - 4:25
QBoC 364 Tu, Th 12:30 - 3:30
VASCI 290F 368 M 1:00 - 3:45
CM&BL 368 W 1:25 - 4:25
CM&BL 360 F 1:25 - 4:25
Histology 264 Tu, W 1:25 - 4:25
Room AM/PM Mon Tues Wed Thurs Fri
360 AM
PM Bioimage 1:25 - 4:25 Model Syst 1:25 - 4:25 Bioimage 1:25 - 4:25 CM&BL 12:20 - 2:30
364 AM
PM QBoC 12:20 - 3:30 QBoC 12:20 - 3:30
368 AM
PM VASCI 290F 1:00 - 3:30 CM&BL 12:20 - 4:30 Model Syst 1:25 - 4:25

2016 Fall

2016 Fall kdorfman Mon, 08/29/2016 - 21:48
Room dept number name day & time instructor TA
264 AnSci 365 Fund lab tech M 1:25 - 3:30 Becker (?)
264 Bio 284 Genetics lab Th 8-12, 1-5 Loomis Angelou
360 Bio 477H Bioimaging MW 1:25-4:25 Wadsworth Estes
360 Bio 397MC Cell & Molec bio lab Th 1:25-4:25 Bezanilla Bascom
364 AnSci 220 Anat & Phys MW 1:25-5:45 Cousin
364 Bio 190H QBoC TuTh 1-4 Rounds Zimmerman
368 AnSci 455 Res An Mgmt TuTh 8:30-11:30 Balise
368 Bio 397MC Cell & Molec bio lab Tu 1:25-4:25 Bezanilla Bascom

2022 Fall

2022 Fall kdorfman Mon, 08/08/2022 - 18:55
Dept course # Prof TA Room Day Time
Bio 284 (genetics) Laney 264 M,W 1:25 - 5:25
Bio 284 (genetics) Loomis 264 Tu,Th 1 - 5
AnSci 220 (A&P) Cousin 364 M,W 11:15 - 5:45
AnSci 366 (Micro) Becker 364 Tu,Th 8:30 - 11:15
AnSci 366 (Micro) Becker 364 F 8 - 10:45
Bio 161H (QBoC) Francis 364 Tu,Th 1-4
AnSci 365 (vet lab techniques) Becker 368 Tu,Th 1-4
AnSci 368 (canine cancer) Arcaro 368 Th 8:30 - 11:15
AnSci 368 (canine cancer) Arcaro 368 W 1 - 3:45
Bio 499CB (hon) Loomis 368 F 9:05 - 12:05
Bio 477H (bioimaging) Stephens Bahiru 360 Tu,Th 1-4
Bio 383H (gene & genome) Maresca 360 M 12:20 - 4:25
Bio 383H (gene & genome) Maresca 360 W 1:25 - 4:25

2024 spring

2024 spring kdorfman Tue, 11/21/2023 - 15:14

Calendars

360 S2024

360 S2024 kdorfman Tue, 11/21/2023 - 15:15

264 S2024

264 S2024 kdorfman Tue, 11/21/2023 - 15:16
Dept no. days time instructor subject materials and equipment
Bio 162 H Tu Th 8:15-11:15 Riley quantitative systems biol microbiology, scopes
Bio 523 Tu
W		 1-4
1:25-4:25		 Spracklen histology scopes, staining
Psych 430 M 9-12 Davidson neuroscience scopes, dissecting
Psych 618 W 10-12 Moorman cognitive science

360 S2024

360 S2024 kdorfman Tue, 11/21/2023 - 15:16
Dept # day time prof subject materials and equipment
Psych 430 M 9-12 Bergan neurosci fl scopes, dissecting, stain
Bio 424 Tu Th 9-12 Okusu marine bio fl , dis scopes, growth chamber
F 9-12
1-4
Bio 477H Tu Th 1-4 Wadsworth Bioimaging Fl scopes, tissue culture

364 S2024

364 S2024 kdorfman Tue, 11/21/2023 - 15:17
Dept # day time instructor subject materials & equipment
Bio 284 Tu th 1-5 Chalufiya genetics lab molecular bio, scopes, incubator
AnSci 366 M
W		 1:25-4:25
11:15 - 2		 Gueye
Becker		 microbiology scopes, incubator
Bio 427 W 2:30 - 5:30 Bartlett perfect plant
AnSci 487 Tu Th 8:30-11:15 Cousin
Alfandari		 embryology dissecting scope, refrigerating incubator

368 S2024

368 S2024 kdorfman Tue, 11/21/2023 - 15:17
Dept # day time instructor subject materials and equipment
AnSci 521 Tu
F		 8:30-11:15
11:15 - 1:25		 reproduction

Labels

Labels kdorfman Wed, 06/17/2009 - 14:15

These are files for making perforated cardstock labels for the frames in the drawers and cupboards, tough tags for 1.5 mL and 0.5 mL microfuge tubes, and tough spots for 1.5 mL microfuge tubes.

You may save any of these files to the desktop and alter it there, or alter it and print it, but you may not save changes to the original.

The labels already written are for illustrative purposes - clear them or rewrite them as you see fit.

anti-tubulin-half-mL2019.docx

0.5 mL microtube labels

0.5 mL microtube labels kdorfman Wed, 06/17/2009 - 14:35

Use this template to make labels for half-mL microfuge labels on Tough-Tags TTSW-2240 from Diversified Biotech (can be ordered from USA Scientific or Krackeler)

The printing tends to drift as the printer moves down the page, so stay away from the margins of the labels.

You can send a page through the printer more than once, but be sure to follow the diagram on the paper feed that shows you which way the paper faces!

half-mL-microtube-labels.doc

1.5 mL microtube labels

1.5 mL microtube labels kdorfman Wed, 06/26/2013 - 20:37
2ml-labels-blank.doc

Brother P-touch 55

Brother P-touch 55 kdorfman Fri, 08/25/2017 - 16:50
p-touch55.pdf

Drawer labels

Drawer labels kdorfman Wed, 06/17/2009 - 14:22

Use this template to make labels for the little frames on the drawers and cupboards.

Print onto the perforated cardstock in the printer drawer in the lab. Use the hand feed paper tray on the front of the printer. You may have to put a stack of paper under the cardstock in order to get the sensor to recognize that this tray has paper in it.

drawer-label-template.doc

Slide Labels

Slide Labels kdorfman Wed, 08/19/2009 - 17:15

These are 22 mm square labels for microscope slides.

9164-1000 from USA Scientific

Make sure you leave enough room on the slide for the label.

Slide-Labels.doc

Spot Labels

Spot Labels kdorfman Wed, 01/27/2010 - 14:33

DFS Item Usa Scientific Plastics Laser Tough-Spots large

Laser Tough-Spots,large,1/2 in. diameter,Labels/Sheet:192,Labels/Package:3840,available in sheets that permanently accept laser printing,Heat-resistant sheets stay flat and will not jam in laser printers,White only

These are for tough-spots to fit the tops of microfuge tubes

Tough spots 1/2 inch Laser sheets: DFS Item Research Products International Corp Tough-Spots Labels > 1/2 Inch Tough-Spots (Labels ) 1000/PKG 1/2 in. diameter Color: yellow Pre-cut round labels Fit 0.5 2.0mL micro-tube caps Withstand autoclaving and liquid nitrogen 1/2 Inch Tough-spots NC9885027 Research Products International Corp No.:247129Y Pack of 1000 for $37.50

spot labels template.doc

Misc supplies

Misc supplies kdorfman Mon, 12/16/2013 - 19:11

Sterile individually wrapped transfer pipets from Krackeler:

119-137135-CS Transfer Polyethylene Built In Bulb Sterile, Size Range 3 to 4mL, Krackeler Value Brand, $21.06 case of 400.

http://www.specialty-graphics.com/cling_film_for_laser_printers_copiers…

EH&S supplies through CEMS

spore test kit: Prospores Mesa Log size 5

Glass beads to spread liquid culture on agar: Fisher 11-312A

Labcoats

Labcoats kdorfman Mon, 11/28/2022 - 14:39

Disposable lab coats

Kept in 264

size color Fisher # mfr #
S cranberry Fisher 23900512A ValueMax 3660CYS
M purple Fisher 23-900-514B ValueMax 3660PPM
L teal Fisher 23-900-511C ValueMax 3660TEL
XL purple Fisher 22770157 (Apex sub for VaueMax) ValueMax 3660PPXL

ValueMax is discontinued

NAP lab

NAP lab kdorfman Fri, 01/14/2022 - 18:42
dates materials
2/1 & 2/3 Microscope boot camp
2/8 students treat embryos with drugs
3/29 projects:
Haloperidol
IWP2
growth hormone
β17-Estradiol
Sodium valproate
Lithium chloride

Drugs:

Orders

Orders kdorfman Tue, 08/13/2019 - 17:53

Billing Address:
UMass Accounts Payable Controllers Office
405 Goodell Building
140 Hicks Way
Amherst, MA 01003

Order spreadsheet up to May 2022

Order Spreadsheet after May 2022 (In Folder: ISB Biolabs Business)

Updating the Order Spreadsheet

  1. Enter any OneCard orders Manually
  2. Export Your Orders from UMASS Buyways
  • Sign into your Buyways Account using your NetID

  • Open a Listing of your Orders

    • Click the "Search" Icon on the far Left (looks like a piece of Paper)
    • Select "My Orders"
    • Select "My Purchase Orders"

  • Organize your List of Orders

    • you may narrow down the results to a specific time frame.
    • You can filter the results as desired
    • you may choose to click to select only specific orders.

*Click "Export All" (use the Drop Down Arrow to select options) (see small Blue Text at the Top Right under "Logout")

  • Create a Title for your Export Request.
    • (e.g.: 2022_10_21 ISB Biolabs Purchases - Packard)
  • Enter the "Type" as: User Defined Template
  • the format will be a csv file
  • Choose Template --> "ISB Biolabs Exporting POs - OFFICIAL Template 10/2022"

  • Click "Submit"

    • In a pop up window you will see and select " Manage Search Exports"
    • Click "Refresh" until your file is generated, this is over to the Right.
    • Copy your results into the Master Orders Spreadsheet on OneDrive

Ethanol orders

Ethanol orders kdorfman Fri, 10/23/2020 - 16:13

Create a new cart

Now go to Fisher punch out. Order ethanol and other chemicals (not non-chemicals or they will go through CEMS delivery).

how many Fisher part # concentration price
1 case/4 each 1 gallon 04355226 190 Proof ethanol $42.90
1 each/5 gallon 04355221 190 Proof ethanol $46.03
1 case/4 each 1gallon 04355223 200 Proof ethanol $42.90
1 each/5 gallon 04355224 200 Proof ethanol $47.29

For alcohol orders, set Commodity Code to Alcohol

In accounting codes tab, make sure accounting code is 739660-A (Tax Free Alcohol)

Check Ship-to address is Chemical Receiving

Assign to your purchaser or Submit Requisition

Change your default shipping address back to normal if you’re ordering non-chemical supplies.

Outreach

Outreach kdorfman Sat, 12/05/2015 - 14:15
fluorescence_cell_bio_summer_2017_.pptx

2022 Eureka

2022 Eureka kdorfman Thu, 07/14/2022 - 19:27

color mixing workshop LLCPK GFP-alpha tubulin cells (have a lot of vacuoles, though)

Made up 2x staining solutions in Fluorbrite:

Nuc blue: 20 uL/2 mL; 40 uL/2 mL

Mitotracker red: 250 nM, 500 nM

Cells incubated in coverslip dish 1 mL nuc blue staining solution + 1 mL mitotracker staining solution.

Hi nuc + Hi MT
Hi nuc + low MT
low nuc + Hi MT
low nuc + low MT

Warm all media first, then remove the F10-Hams, replace with appropriate staining solutions. Keep cells in incubator for 15 min, check...

Color Mixing with Inks CMY(K)

1:15 (approximate) yellow printer ink in water 1:200 magenta 1:200 cyan

we pipetted various ink amounts onto parafilm to mix colors - final volume of droplets = 100ul

Additive Mixing with Light (RGB Flashlights) Flashlights made by RaySoar LED Limited Company

Staining solution NucBlue + Mitotracker

Staining solution NucBlue + Mitotracker kdorfman Fri, 09/30/2022 - 13:47

Staining Solution

  • 1 mL fluorobrite
  • 10uL NucBlue
  • 1 uL Mitotracker red

Staining Protocol

  • Remove pink medium from cells (~2.5 mL)
  • Replace with Staining Solution
  • Rock dish to make sure solution covers the surface
  • Incubate ~15 min at 37C

Observation Protocol

  • Find cells with phase
  • Switch to fluorescence
    • turn on arc lamp
    • turn off white light
    • open mechanical shutter (CLOSE WHEN NOT LOOKING AT CELLS! THEY BLEACH!)
    • open electronic shutter
    • turn filter cube to UV to see blue nuclei
    • turn filter cube to G to see red mitochondria

BioBootCamp

BioBootCamp kdorfman Tue, 06/20/2017 - 15:48
2016micropipetting.docx
pipet_buffer_table.docx
double_pipet_buffer_table.docx

2017 bio boot camp

2017 bio boot camp kdorfman Tue, 06/20/2017 - 18:02

24 students

  • pub med search
  • BLAST
  • pipetting
  • Serial dilution
  • calculations
  • DNA extraction (G&GA protocol)
  • Quantification by nanodrop
  • gel electrophoresis - w/ & w/o RNAse
  • wide range marker
umasssummcoll.labbootcampmanual2017.v6.pdf

2022 Bio Boot Camp

2022 Bio Boot Camp kdorfman Wed, 06/22/2022 - 18:20

FIRST DAY

Pipetting Exercise

DNA Extraction

  • Extract DNA from fish fin clips with HotSHOT DNA extraction prep

    • 50 uL alkaline lysis reagent per rxn
    • 50 uL Neutralization buffer per rxn
    • Aliquot 1 mL of each per pair

  • Rolf brings 20 fin clips

  • Students work in pairs, doing 4 clips per pair.

Set up PCR reactions

  • Use Qiagen Taq PCR Master Mix Kit

  • Dilute primers from Rolf's lab for GFP (final 0.4 uM in mix)

  • Each 25 uL reaction contains:
    • 12.5 uL 2X Master mix
    • enough primer to make 0.4 uM
    • 5 uL DNA
    • water to 25 uL
  • We make 900 uL Master Mix with primers
    • 3.6 uL each 100uM primer
    • 450 uL 2x master mix
    • 446.4 uL water (to final volume 900 uL)
  • Each individual student tests all 4 of the DNA samples
    • 20 uL per rxn
    • aliquot 180 uL per pair

Make enough master mix for 9 reactions per pair

PCR program (finclip in Main menu):

Step time (min) temp comment
1. Initial denaturation 2 94
2. denaturation 0.5 94
3. annealing 0.5 55 ~4C below primer Tm
4. extension/elongation 0.5 72 or one min per kb 1
5. go to 2 38 times
6. final extension 5 72
7. hold forever 15 can cut short and put into fridge

SECOND DAY

Gel

BLAST

Use primer sequences?

Analyze Gel

  • check bands in blue light
  • take cell phone photo
  • send to Rolf with documentation

  1. 45 sec is better for mcherry; can use 45 sec to 1 min for GFP. EGFP and mcherry amplicons are ~500 BP ↩︎

Qiagen Taq PCR Master Mix Handbook

Master mix

Master mix kdorfman Mon, 06/12/2023 - 15:05
To make Qiagen Master Mix + primers for students
and reactions per student
at uL per reaction
Start with: uL Qiagen Master Mix
add: uL forward eGFP primer (100 mM)
add: uL reverse eGFP primer (100mM)
add: uL forward Hedgehog Inhibitory Protein primer (100 mM)
add: uL reverse Hedgehog Inhibitory Protein primer (100 mM)
add: uL water
for a final volume of: uL Qiagen Master Mix plus primers
aliquot: uL Qiagen Master Mix plus primers per student

2023 Boot Camp

2023 Boot Camp kdorfman Wed, 06/21/2023 - 18:53
  1. streamlined pipet exercise
  2. Genotyping of Rolf's fish

Zebrafish finclip genotyping

Zebrafish finclip genotyping kdorfman Wed, 06/21/2023 - 18:56

Copied from 2022:

*DNA Extraction**

  • Extract DNA from fish fin clips with HotSHOT DNA extraction prep

    • 50 uL alkaline lysis reagent per rxn
    • 50 uL Neutralization buffer per rxn
    • Aliquot 1 mL of each per pair

  • Rolf brings 20 fin clips

  • Students work in pairs, doing 4 clips per pair.

  • Master mix recipe

Set up PCR reactions

  • Use Qiagen Taq PCR Master Mix Kit

  • Dilute primers from Rolf's lab for GFP (final 0.4 uM in mix)

  • Each 25 uL reaction contains:
    • 12.5 uL 2X Master mix
    • enough primer to make 0.4 uM
    • 5 uL DNA
    • water to 25 uL
  • Karlstrom recipe for 900 uL Master Mix with primers: (see Master mix calculator)
    • 3.6 uL each 100uM primer
    • 450 uL 2x master mix
    • 446.4 uL water (to final volume 900 uL)
  • Each individual student tests all 4 of the DNA samples
    • 20 uL per rxn
    • aliquot 180 uL per pair

Make enough master mix for 9 reactions per pair

PCR program (finclip in Main menu) (don't know which thermocycler)

Thermocycler 3 in 364: folder BIOBOO: programs HOT92, ZFPCR

Step time (min) temp comment
1. Initial denaturation 2 94
2. denaturation 0.5 94
3. annealing 0.5 55 ~4C below primer Tm
4. extension/elongation 0.5 72 or one min per kb 1
5. go to 2 38 times
6. final extension 5 72
7. hold forever 15 can cut short and put into fridge

SECOND DAY

Gel

BLAST

Use primer sequences?

Analyze Gel

  • check bands in blue light
  • take cell phone photo
  • send to Rolf with documentation

  1. 45 sec is better for mcherry; can use 45 sec to 1 min for GFP. EGFP and mcherry amplicons are ~500 BP ↩︎

Eureka 2015

Eureka 2015 kdorfman Thu, 07/30/2015 - 19:13
dendrobium_piercardii_stem_section1.jpg
salvinia_plant9.jpg
salvinia_plant10.jpg
imaris_hair4.jpg
plant1.jpg
plant2.jpg
sabal_minor_plant3.jpg
adromischos_root_section7.jpg
adromischos_root_section8.jpg
adromischos_stomata3.jpg
adromischos_stomata4.jpg
adromischos_stomata5.jpg
adromischos_vascular_bundles6.jpg
my_plant_22.jpg
salvinia_plant8.jpg
salvinia_plant7.jpg
heliania1.jpg
strelitzia_reginae1.jpg
strelitzia_reginae2.jpg
strelitzia_reginae3.jpg
strelitzia_reginae4.jpg
easypeel_ic1.jpg
zamia_furfuraceae_01_ic2.jpg
salvinia_plant1.jpg
salvinia_plant2.jpg
salvinia_plant3.jpg
salvinia_plant4.jpg
salvinia_plant5.jpg
salvinia_plant6.jpg
my_plant_1.jpg

MassBio

MassBio kdorfman Sat, 12/05/2015 - 14:16

DIY

DIY kdorfman Sat, 12/05/2015 - 14:20
do_it_yourself_biotech_equipment_and_paper_lab_workshop_agenda_winter_2015.docx
the_tennessean.pages_.pdf

Science Quest 2014

Science Quest 2014 kdorfman Tue, 04/08/2014 - 18:24
Participant Status major course project
Shelley Kratzer sr bio 499F actin?
Vishakha Agrawal sr bio/psych 383H MW genotyping
Dylan Bennet bio 383H MW genotyping
Jenna McMahon jr bio 383H TuTh
Heather Jordan sr bio 499F actin?

Pipettes and tips

Pipettes and tips kdorfman Thu, 06/18/2009 - 16:04

pipette inventory

https://docs.google.com/spreadsheets/d/1aKkA5NNv_3jWn3p5e2WyZPCDCPmfvoy…

pipet_buffer_table.docx

Classroom pipettes

Classroom pipettes kdorfman Thu, 01/03/2013 - 17:41

The pipettes in use in the ISB are Rainin micropipettors :

  • Pipet-Lite® with LTS®, which takes LTS cylindrical tips, and has red, green, or blue labels on the plunger

LTS PIPETTES (rooms 260, 262A, 268, 360, 364, 366A, 368)

TIPS

Plunger max uL part #
Blue 1000 Rainin GPS-LTS 1000uL tips (30389292), in GPR-L1000 (blue) racks
Green 200 Rainin GPS-LTS 250 uL tips (30389299), in GPR-L250 (green) racks
Red 20 Rainin GPS-LTS 20uL tips (30389291), in GPR-L10 (red) racks
  • A 1-12 (264)
  • B 1-12 (368)
  • C 1-12 (364)
  • D 1-11 (360)
  • E 1-5 (371)

Colorimetric Pipetting Exercise

Colorimetric Pipetting Exercise kdorfman Wed, 06/21/2023 - 18:16

Mix 200 mM mono- (acid) and di-basic sodium phosphate (base) with bromothymol blue to get a range of colors from yellow to blue.

Put a 5 uL drop of bromothymol blue in each square, then add the given amounts of sodium phosphate

pH 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8
Na2HPO4 2.5 3.7 5.3 7.5 9.8 12.2 14.4 16.2 17.4 18.3
NaH2PO4 17.5 16.3 14.7 12.5 10.2 7.8 5.6 3.8 2.6 1.7

Per exercise, need:

  • 107.3 uL Na2HPO4
  • 92.7 uL NaH2PO4
  • 50 uL bromothymol blue (0.04% aqueous) (e.g., Fisher LC120501, $25 for 500 mL))
  • printed buffer table (see linked file)
  • parafilm

So aliquot

reagent actual vol aliquot
Na2HPO4 107.3 250
NaH2PO4 92.7 250
bromothymol blue 50 125
pipet buffer table

Eppendorf Repeater

Eppendorf Repeater kdorfman Mon, 09/04/2023 - 20:17

Use Combitips Advanced:

Dial # aliquots 0.1 mL 0.2 mL 0.5 mL 1.0 mL 2.5 mL 5.0 mL 10 mL 25 mL 50 mL
white lt blue purple yellow green blue orange red gray
µL µL µL µL µL µL mL mL mL
* 100 1 2 5 10 25 50 0.1 0.25 0.5
1 50 2 4 10 20 50 100 0.2 0.50 1
* 33 3 6 15 30 75 150 0.3 0.75 1.5
2 25 4 8 20 40 100 200 0.4 1.00 2
* 20 5 10 25 50 125 250 0.5 1.25 2.5
3 16 6 12 30 60 150 300 0.6 1.50 3
* 14 7 14 35 70 175 350 0.7 1.75 3.5
4 12 8 16 40 80 200 400 0.8 2.00 4
* 11 9 18 45 90 225 450 0.9 2.25 4.5
5 10 10 20 50 100 250 500 1 2.50 5
* 9 11 22 55 110 275 550 1.1 2.75 5.5
6 8 12 24 60 120 300 600 1.2 3.00 6
* 7 13 26 65 130 325 650 1.3 3.25 6.5
7 7 14 28 70 140 350 700 1.4 3.50 7
* 6 15 30 75 150 375 750 1.5 3.75 7.5
8 6 16 32 80 160 400 800 1.6 4.00 8
* 5 17 34 85 170 425 850 1.7 4.25 8.5
9 5 18 36 90 180 450 900 1.8 4.50 9
* 5 19 38 95 190 475 950 1.9 4.75 9.5
10 5 20 40 100 200 500 1000 2 5.00 10
printable table

Prep Room Pipettes

Prep Room Pipettes kdorfman Thu, 01/03/2013 - 18:51

Rainin

Shop Rainin

F1 (362A)

  • 10 mL
  • 5 mL
  • 1 mL
  • 200 uL
  • 20 uL
  • 10 uL

F2 ( 266A)

  • 5 mL
  • 1 mL
  • 200 uL
  • 20 uL
  • 10 uL

F3 (donated to Animal Science)

5 mL pipette SL-5000XLS
Tips: LTS 5 mL 192/8 RT-L5000
Item # 17002937
Rainin Hinged Racks Box reads 30389256

10 mL pipette
Tips LTS 10 mL Prstrl 75/Pkg RC-L10MLS pre-sterilized, individually wrapped
Item #17005940

Eppendorf Repeater Plus in 362

Combitips Advanced

Get prices on tips from Krackeler

Protocols

Protocols kdorfman Tue, 06/16/2009 - 20:02

How to do stuff

How to get containers and stickers from EH&S

Sterilization

Sterilization kdorfman Tue, 06/16/2009 - 17:50

Sterilize anything that cells might grow in.

Autoclave glassware, tips, and microfuge tubes.

Autoclave most media and salt solutions.

Do not sterilize by autoclave items containing:

  • detergents (e.g., SDS) - they can boil over

  • heat sensitive ingredients (e.g., vitamins, hormones, antibiotics, proteins)

  • sugar in growth medium (the sugars and amino acids may react together, reducing the concentration of both)

  • HEPES

  • DTT (dithiothreitol)

  • Beta mercaptoethanol

  • corrosives (e.g. acids, bases, phenol)

  • solvents or volatiles (e.g. ethanol, methanol, chloroform, acetone, formaldehyde, formalin or glutaraldehyde)

  • chlorine (e.g., bleach)

  • anything radioactive

Filter sterilize any liquid that must be sterile, and that you cannot autoclave.

biohazard_waste_only.docx

Autoclave

Autoclave kdorfman Tue, 06/16/2009 - 20:25

autoclave manual

Always put a piece of autoclave tape on the item or container so you can tell if it was exposed to the steam.

If the green light is on, press the red reset button.

  • Biohazard Waste: See here: https://wahoo.nsm.umass.edu/content/biohazard-waste

  • Tips: Load racks into boxes wearing gloves (this caution is primarily for RNA work, as most people's skin has RNase on it).
    15 minute sterilization; 40 min drying time; open autoclave CAUTION - HOT! to let steam escape.
    If there is too much condensation inside the boxes, put them in the oven at ~60C for ~an hour.

  • Microtubes: Put into 600 mL plastic jars, screw cover on loosely, so the steam will penetrate. Same time as for tips.

  • Liquids: Larger volumes require longer sterilizing times. Use this table:

Largest volume (mL) Minimum time (min)
75 25
250 30
500 40
1000 45
1500 50
2000 55
>2000 55 + 10 per L
  • Monthly spore test EH&S recommends Fisher 12-001-1 population 10^5 Prospore Bacillus stearothermophilus
To sterilize mL of liquid medium
autoclave for: minutes

Minutes = 8.3853 * volume^0.2449

autoclave_log.docx
autoclavemanual.pdf

Monthly Maintenance

Monthly Maintenance kdorfman Fri, 09/09/2016 - 15:45

  • Turn generator switch off

  • Let cool to ~5 lb pressure

  • Turn master switch off

  • Open valve

  • Let tank drain

  • Turn generator switch on

  • Close valve

  • Let it fill

  • Turn master switch on

Autoclave bags

Autoclave bags bcrcstaff Thu, 02/14/2019 - 13:45
Container dimensions (in) bag size
Rubbermaid 16" x 10" 24" x 36" Fisher 01-8143
Round diam = 18" 38" x 48" VWR 14220-044
Square 16" x 14" 38" x 48" VWR 14220-044

Dishwashing

Dishwashing kdorfman Tue, 06/30/2009 - 20:35

There are dishwashers in rooms 261 and 361.

Instructions are in the drawer labeled "manuals" in each room, and linked to this page. "Dishwashing" gives general user instructions; "Dishwash-program-guide" explains how to change the cycles - for advanced users only.

Replacement detergent: Fisher 04-319B Thermo Scientific* Nalgene* L900 Liquid Detergent

1 gal $59

4 gal $167 (@$42)

Dishwashing.pdf
Dishwash-program-guide.pdf

Drierite Regeneration

Drierite Regeneration margaret Thu, 10/06/2011 - 18:34

For crystals: 1 hour at 210° C (=425° F) in shallow glass pan. See details below:

For cartridge: 3 hours at 150° C (=~300° F), perforated top down. Blue means full reactivation.

REGENERATION OF DRIERITE DESICCANTS After normal use, any of the forms of DRIERITE may be regenerated for reuse. The operation is simple and involves only standard equipment. The used and exhausted desiccant should be ventilated to remove vapors, if any, and stored in a convenient container until a sufficient amount is accumulated to justify the work of regeneration.

Regular and Indicating DRIERITE For the regeneration of Indicating DRIERITE and small lots of Regular DRIERITE , the granules may be spread in layers one granule deep and heated for 1 hour at 210° C or 425° F. The regenerated material should be placed in the the original glass or metal container and sealed while hot. The color of the Indicating DRIERITE may become less distinct on successive regenerations due to the migration of the indicator into the interior of the granule and sublimation of the indicator.

The Importance of Temperature The temperature at which DRIERITE desiccants are regenerated is crucial in restoring DRIERITE to its original condition. Absorbed moisture is water of hydration and is chemically bound to the calcium sulfate of DRIERITE. Temperatures in the range of 400° - 450° F are required to break these bonds and release absorbed moisture. Lower temperatures, regardless of heating time, will not regenerate DRIERITE unless applied under vacuum (28" Hg, 325° F or 26" Hg, 275° F). Care should be taken not to overheat DRIERITE Desiccants. High temperatures can alter the crystal structure and render the desiccants permanently inactive.

Mini-prep

Mini-prep kdorfman Thu, 09/15/2022 - 15:33

Keep stocks of 2 mini-prep kits:

Qiagen

Zymo Classic D4015

Qiagen

Qiagen kdorfman Thu, 09/15/2022 - 19:53

Jeff's CMBL miniprep

Zymo miniprep classic

Zymo miniprep classic kdorfman Thu, 09/15/2022 - 19:53

For many labs

Before beginning, add 95% ethanol to Plasmid Wash Buffer 4:1 Also, once RNAse has been added to P3 (yellow), keep it at 4C.

Protocol

  • Spin 0.5 - 5 mL bacterial culture in 1.5 mL tube 20 sec full speed

  • Add 200 uL P1 (red), resuspend pellet

  • Add 200 uL P2 (green); mix by inverting 2-4 times. Clear, viscous, purple solution indicates cell lysis

  • Add 350 uL P3 (yellow). Mix thoroughly. DO NOT VORTEX. Turns yellow when neutralization is complete.

  • Incubate RT 1-2 min

  • Spin 2 min

  • Put column into collection tube, transfer supernatant. DO NOT DISTURB GREEN PELLET (I found it wasn't green, but OK)

  • Spin 30 sec

  • Discard flow through, put column back into emptied collection tube

  • Add 200 uL Endo-Wash Buffer to the column, spin 30 sec

  • Add 400 uL Plasmid wash buffer to the column, centrifuge 1 min

  • Transfer column to clean 1.5 mL microcentrifuge tube

  • Add 30 uL DNA Elution Buffer

  • Spin 30 seconds. Plasmid DNA is in the collection tube

Students need per reaction1

  • 1 mL (?) overnight culture of transformed cells

  • 1 column, 1 2-mL collection tube

  • sterile 1.5 mL tube

  • ~200 uL P1 (red)

  • ~200 uL P2 (green)

  • ~350 uL P3 (yellow)

  • ~200 uL Endo-Wash-Buffer

  • ~400 Plasmid Wash Buffer

  • ~30 uL DNA Elution Buffer

  1. All the ingredients are sold separately, so it's OK to give students some extra to allow for pipetting errors. ↩︎

ZR Plasmid Miniprep Classic

Peroxide testing

Peroxide testing kdorfman Tue, 10/09/2018 - 20:46

Preparation

  • Samples containing more than 25 mg/L H2O2 (Cat. Nos. 110011) or 100 mg/Ll H2O2 (Cat. No.110081) must be diluted with distilled water or peroxide-free ether.

  • The pH of the aqueous sample must be within the range 2-12 If necessary, buffer the sample wit sodium acetate or, respectively, adjust the pH with hydrochloric acid

Test Procedure

  • For aqueous solutions:

    • Immerse the reaction zone of the test strip in the pretreated sample (15 – 30 C) for 1 sec.
    • Allow excess liquid to run off via the long edge of the strip onto an absorbent paper towel and after 15 sec (Cat. No. 110011) or after 5 sec (Cat. No. 110081) determine with which color field on the label the color of the reaction zone coincides most exactly.
    • Read off the corresponding result in mg/L H2O2

  • For organic solvents:

    • Immerse the reaction zone of the test strip in the pretreated sample (15 – 30 oC) for 1 sec.

    • After the solvent has evaporated (gently fan the strip back and forth for 3 – 30 sec), immerse the reaction zone in distilled water for 1 sec and allow excess liquid to run off via the long edge of the strip onto an absorbent paper towel.

    • After 15 sec (Cat. No. 110011) or after 5 sec (Cat. No. 110081) determine with which color field on the label the color of the reaction zone coincides most exactly.

    • Read off the corresponding result in mg/L H2O2

General Notes

  • any blue within 3 minutes is a positive result

  • If the color of the reaction zone is equal to or more intense than the darkest color on the scale or in another color emerges, repeat the measurement using fresh samples diluted with distilled water or, respectively, peroxide-free ether until a value of less than 25 mg/L/ H2O2 (Cat. No. 110011) or 100 mg/L (H2O2) (Cat. No. 110081) is obtained.

In the case of Cat. No. 110081 the reaction zone indicates values within the measuring range also for H2O2 contents from 5000 mg/L (0.5 %) up.

peroxide_forming_material_handout2018.pdf

Weekly Inspections

Weekly Inspections kdorfman Tue, 10/11/2011 - 16:47

Biohazard Waste

Biohazard Waste kdorfman Tue, 10/11/2011 - 16:50

Check the red biohazard trashcans, usually found in 360, 364, or 368.

If it is more than half full, or if it really stinks, autoclave it.

Wear gloves! Put the clear bag into a Nalgene autoclave basket, but don't seal it up tight. (If you do, it will explode in the autoclave.) Double bag it if it has a lot of liquid in it.

Set it for 60 minutes sterilization, liquid cycle.

When it is done, make sure the "autoclaved" sign on the bag has turned dark, then seal the bag and put a non-hazardous waste sticker on it, put it inside a black trashbag, and throw in the regular trash.

Recipes

Recipes kdorfman Tue, 06/16/2009 - 20:04

Antibodies

Antibodies kdorfman Tue, 10/02/2018 - 16:21

For initial dilution of lyophilized antibody:

  • Dissolve to recommended concentration (per package insert) with half glycerol and half water.

  • Aliquot and freeze.

  • Label the tubes or the box with how much to dilute.

Primary Antibodies

Primary Antibodies kdorfman Tue, 10/02/2018 - 16:21

5-mC (rabbit)

5-mC (rabbit) kdorfman Fri, 11/05/2021 - 13:44

5-methylcytosine (D3S2Z)

Rabbit mAb

Binds to methylated cytosine

28692S Cell Signaling Technology

From the manufacturer: "Methylation of DNA at cytosine residues is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting, and mammalian development (1,2). 5-methylcytosine is a repressive epigenetic mark established de novo by two enzymes, DNMT3a and DNMT3b, and is maintained by DNMT1

Dilute 1:1600

Before Antibody treatment, treat fixed cells with 4N HCl for 15 minutes to denature DNA.

Neutralize with 2 washes with sodium borate pH 9, then PBS. Then start the PBS-Tw-Az rinses before incubating with the antibody.

Store at -20

Actin (mouse)

Actin (mouse) kdorfman Tue, 10/02/2018 - 18:50

DSHB JLA20

stock concentration = 22 ug/mL

working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 90 uL ~11 mg/mL

Add 216 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

Actin (rabbit)

Actin (rabbit) kdorfman Tue, 10/02/2018 - 19:04

Sigma A2668

Dilute 1:40 for use

Dilute 1:1 with glycerol

Aliquot 16 uL

Add 290 uL PBS BSA to make enough for 3 slides

Freezer 266A

a2668dat-1.pdf

Alkaline phosphatase (mouse)

Alkaline phosphatase (mouse) kdorfman Tue, 10/02/2018 - 18:51

DSHB B4-78

stock concentration = 38 ug/mL

diluted 1:1 in glycerol

50 uL aliquots of ~19ug/mL

Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides

266A freezer

Alpha-actinin (mouse)

Alpha-actinin (mouse) kdorfman Tue, 10/02/2018 - 17:46

Binds actin to membrane at adherin junctions
Mouse monoclonal, Clone BM 75.2
IgM
Fix in formaldehyde (no MeOH or glutaraldehyde)
1:200
Use anti-mouse IgM FITC secondary

Cadherin (mouse)

Cadherin (mouse) kdorfman Tue, 10/02/2018 - 17:37

"calcium-dependent adhesion"

cell adhesion molecule (CAM) important in formation of adherens junctions to bind cells with each other.

Class of type-1 transmembrane proteins.

Dependent on calcium (Ca2+) ions to function

small aliquot: 5 µL
large aliquot: 10 µL

Collagen II

Collagen II kdorfman Tue, 10/02/2018 - 18:59

DSHB II-II6B3

stock solution: 96 ug/mL

working solution: 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 20 uL ~48 mg/mL

Add 286 uL PBS-Tw-BSA to make enough for 3 slides

266A Freezer

Collagen IV

Collagen IV kdorfman Tue, 10/02/2018 - 19:00

DSHB M3F7

mouse anti collagen IV (basement membranes)

2 tubes as of spring 2018:

  • stock concentration (8/7/08) = 30 ug/mL
  • stock concentration (12/22/11) = 39 ug/mL

working concentration = 2 - 5 ug/mL

Each diluted with glycerol to a concentration of 18.5 ug/mL

Aliquots are 50 uL

Add 256 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

Connexin (rat)

Connexin (rat) kdorfman Tue, 10/02/2018 - 19:17

DSHB R5.21C

stock concentration = 28 ug/mL

working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 70 uL ~14 mg/mL

Add 236 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

DNMT1 (rabbit)

DNMT1 (rabbit) kdorfman Fri, 11/05/2021 - 13:39

D63A6 XP(R)

Rabbit mAb

5032S Cell Signalling Technology

detects endogenous levels of total DNMT1 protein.

From the manufacturer: "Methylation of DNA at cytosine residues in mammalian cells is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting and development (1,2). Three families of mammalian DNA methyltransferases have been identified: DNMT1, DNMT2 and DNMT3 (1,2). DNMT1 is constitutively expressed in proliferating cells and functions as a maintenance methyltransferase, transferring proper methylation patterns to newly synthesized DNA during replication."

Dilute 1:100 for use

Golgi (mouse)

Golgi (mouse) kdorfman Tue, 10/02/2018 - 17:11

1 µL aliquots in tube. Store in freezer (-20C)

add 100 µL 1% BSA in PBS

Use just under 50 µL per coverslip (so there is enough for 2 per tube)

[edited 9/26/18 - used to say 1:200 dilution, but we had better results with 1:100]

Hec1 (mouse)

Hec1 (mouse) kdorfman Tue, 10/02/2018 - 16:23

Novus Biologicals, recombinant human Hec1, monocolonal
fix in methanol
dilute 1:200

Histone H3 phospho S10

Histone H3 phospho S10 kdorfman Fri, 11/04/2022 - 20:46

FINISH WORKING ON THIS!

Rabbit polyclonal

Abcam AB5176

1 mg/mL, 100 ug, should be 100 uL in the package

1:2000

Storage:

  • Dilute 1;10 glycerol
  • 2 uL aliquots; for use, raise to 400 uL
  • leave the rest with a label: dilute 1 uL in 200 uL PBS-Tw-Az-BSA

Integrin (rat)

Integrin (rat) kdorfman Tue, 10/02/2018 - 19:18

DSHB Ralph 3.1

rat anti-integrin-3

IgG2a

stock concentration = 27 ug/mL

working concentration = 2 - 5 ug/mL

Freezer 266A

LAMP1 (lysosomes) (mouse)

LAMP1 (lysosomes) (mouse) kdorfman Tue, 10/02/2018 - 16:22

GeneTex GTX13523

IgG

1 mg/mL

4C short term

Freeze 1 µL aliquots

dilute 1:100 - 1:750 in 0.1% BSA in PBS

rinse in PBS

Fix

1% BSA in PBS 30 min

1o Ab stain (0.1% BSA) overnight at 4C
wash 30 min in PBS

2o Ab 1 h 37C
wash 30 min PBS

mount in DAPI

reference

LAP2 (mouse)

LAP2 (mouse) kdorfman Tue, 10/02/2018 - 18:48

Stains nuclear envelope

Lamin A/C

Lamin A/C kdorfman Fri, 11/04/2022 - 20:36

Cell Signalling 4777S

mouse monocolonal (4C11)

7 ug/mL

Use at 1:100

(20 uL/class)

Storage:

  • dilute 1:1 with glycerol,
  • make 5 uL aliquots;
  • add 245 uL PBS-Tw-Az-2% BSA

Lamin B1

Lamin B1 kdorfman Tue, 10/17/2023 - 16:30

Rabbit anti-Lamin B1

Abcam AB16048

comes 100 µL at 1 mg/mL

Says to use at 0.1 µg/mL = 1:10,000 Drew's lab uses it at 1:1000

Make aliquots:

  • 2 µL in a 2 mL tube
  • label says
    • 2 µL rabbit anti-lamin B1
    • add 2 mL PBS-Tw-Azide

Aliquot and freeze - do not refreeze

Laminin (mouse)

Laminin (mouse) kdorfman Tue, 10/02/2018 - 19:01

DSHB 2E8

mouse anti-laminin gamma 1

stock concentration = 26 ug/mL

working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 80 uL ~12 mg/mL

Add 226 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

Mucin (mouse)

Mucin (mouse) kdorfman Tue, 10/02/2018 - 19:03

Monoclonal Anti-Mucin Gastric antibody produced in mouse

Sigma M5293

dilute 1:200 for use

dilute 1:1 in glycerol

Aliquot 4 uL; add 302 uL PBS BSA to make enough for 3 slides

Freezer 266A

m5293dat-1.pdf

Myosin (mouse)

Myosin (mouse) kdorfman Tue, 10/02/2018 - 18:46

DSHB T14

mouse anti-myosin light chain

stock solution: 37 ug/mL

working solution: 2 - 5 ug/mL

diluted 1:1 in glycerol

50 uL aliquots of ~18ug/mL

Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides

Freezer 266A

Myosin (rabbit)

Myosin (rabbit) kdorfman Tue, 10/02/2018 - 19:14

Sigma M7523

Formalin fixation

2 vials as of spring 2018

Dilute 1:20

freezer 266A

m7523dat.pdf

Neurofilament (rabbit)

Neurofilament (rabbit) kdorfman Tue, 10/02/2018 - 19:16

Anti-Neurofilament 200 antibody produced in rabbit

Sigma N4142

Dilute 1:80

Freezer 266A

n4142dat.pdf

Nuclear pore complex

Nuclear pore complex kdorfman Wed, 10/18/2023 - 19:16

abcam ab24609

Data Sheet

Anti-Nuclear Pore Complex Proteins antibody (Mab414)

100 µL, 1 mg/mL

Dilute 1:1000 to use

2 µL aliquots. Label says:

  • add 198 µL PBS-Tw-Az-BSA
  • dilute again 1:10 for use

Mouse monoclonal [Mab414] to nuclear pore complex proteins

Suitable for ICC or IF

Reacts with mouse, human, Saccharomyces cerevesiae

Isotype: IgG

Store at 4C short term

Aliquot and freeze for long term storage

Fix in 100% methanol -20C OR 4% PFA in PBS pH 7.4 10 min RT

24609 data sheet

PKC (rabbit)

PKC (rabbit) kdorfman Tue, 10/02/2018 - 20:44

PKC alpha (c-20)

rabbit polyclonal Abs 1gG

DO NOT FREEZE

In refrigerator 36sA

dilute 1:50 to use

Santa Cruz Biotechnology sc-208

From Wikipedia: Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control. Knockout studies in mice suggest that this kinase may be a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes.[5]

Protein kinase C-alpha (PKC-α) is a specific member of the protein kinase family. These enzymes are characterized by their ability to add a phosphate group to other proteins, thus changing their function. PKC-α has been widely studied in the tissues of many organisms including drosophila, xenopus, cow, dog, chicken, human, monkey, mouse, pig, and rabbit. Many studies are currently being conducted investigating the structure, function, and regulation of this enzyme. The most recent investigations concerning this enzyme include its general regulation, hepatic function, and cardiac function.

Synaptic vesicle (mouse)

Synaptic vesicle (mouse) kdorfman Tue, 10/02/2018 - 18:57

DSHB SV2

Synaptic vesicle glycoprotein 2A

Stock concentration = 36 ug/mL

Working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 60 uL ~18 mg/mL

Add 246 uL PBS-Tw-BSA to make enough for 3 slides

Freezer 266A

Troponin (mouse)

Troponin (mouse) kdorfman Tue, 10/02/2018 - 18:58

DSHB TI-4-s

Anti-troponin I

IgG (mouse)

Stock concentration = 43 ug/mL

Working concentration = 2 - 5 ug/mL

Diluted 1:1 with glycerol

Aliquots are 50 uL ~22 mg/mL

Add 256 uL PBS-Tw-BSA to make enough for 3 slides

266A Freezer

Tubulin alpha (rat)

Tubulin alpha (rat) kdorfman Tue, 10/02/2018 - 16:23

Accurate Chem YSRTmcADg YSRTMCA77G

Clone YL 1/2

Antibody as purchased (2019) was diluted 1:1 with glycerol

As purchased, should be diluted 1:200; glycerol-diluted AB should be diluted 1:100 with PBS-Tw-Az-BSA

Each tube has 4 µL antibody

Add:
400 µL PBS-Tw-Az-BSA =404 µL, enough for 8 reactions (50 µL per coverslip, 60 min 37C)

Incubate coverslip on 50 uL antibody 1 hr 37C in humid chamber

Tubulin gamma (mouse)

Tubulin gamma (mouse) kdorfman Tue, 10/02/2018 - 17:17

binds to centrosomes

From Pat
Monoclonal, Sigma clone GTU-88
Fix in paraformaldehyde or cold methanol
Dilute 1:100

From Drew Sigma T6557-100UL

  • Working concentration is 1:5000
  • Frozen stock is diluted 1:1 with glycerol
  • 5 uL aliquots (in half-mL tubes) should be diluted:
    • 1:50 in the tube: add 245 uL PBS-Tw-BSA to make 1:100 stock
    • 1:50 for use (1 uL 1:100 antibody + 49 uL PBS-Tw-Az-BSA per coverslip)

Drew's Protocol:

  • Rinse in PBS
  • Fix in paraformaldehyde 15 minutes
  • Rinse with PBS-Triton
  • Rinse with PBS-Tween
  • Rinse with PBS-BSA 1 hour
  • Dilute Ab 1:5000 in BSA
  • Incubate with Ab 2hr RT
  • Rinse with PBS 3x
  • Incubate with secondary Ab (in BSA)
  • Rinse with PBS 3x
  • Mount

Test Protocol:

  • coverslips already fixed in paraglut, stored in PBS-Tw-Az 1
  • 1 uL Ab (as supplied by Sigma) into 99 uL PBS-BSA (save!)
  • 1 uL 1:100 Ab into 499 uL PBS-BSA (save for the rest of the slides if it works!)
  • 50 uL onto coverslip - 2 hr RT incubation
  • rinse with PBS-Tw-Az
  • 50 uL Goat anti mouse 488

  1. Results: some background fluorescence. Add a blocking step:
    rinse 30 min in 2% BSA
    rinse 2x in PBS-Tw-Az
    Didn't make much difference. centrosomes are small and bright ↩︎

t6557-bulk_sigma_.pdf
protocol from Stephens lab

Vinculin (mouse)

Vinculin (mouse) kdorfman Tue, 10/02/2018 - 18:44

Membrane-cytoskeletal protein in focal adhesion plaques that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton, associated with cell-cell and cell-matrix junctions, where it is thought to function as one of several interacting proteins involved in anchoring F-actin to the membrane.

Sigma V4505 monoclonal mouse
Fix in Formaldehyde
Dilute 1:100

ZO1 (Rabbit)

ZO1 (Rabbit) kdorfman Tue, 10/02/2018 - 16:24

large tube has 10 µL antibody

small tube has 5 µL antibody

use at 1:100

362A freezer

fix in paraformaldehyde

Secondary antibodies

Secondary antibodies kdorfman Tue, 10/02/2018 - 19:19

Goat anti-mouse IgM TRITC

Goat anti-mouse IgM TRITC kdorfman Tue, 10/02/2018 - 19:26

Fisher OB102102 (Southern BioTech 1021-02)

Goat Anti-Mouse IgM (micron chain specific)-FITC

1 mg/mL

1:100 - 1:400

1 µL aliquots. Add 100 - 400 µL 1% BSA

Use 50 µL per coverslip

sobiotech1021-goat-anti-mouse-igm-2.pdf

Goat anti-mouse 488

Goat anti-mouse 488 kdorfman Tue, 10/02/2018 - 20:37

Invitrogen A-11001

Ex: 488 nm

Em: 499 nm

Stock(as purchased) = 2 mg/mL

Stock bottle has been diluted with glycerol: ~800 uL of 1 mg/mL

Working concentration = 1 ug/mL

Aliquots are 1 uL of 1 mg/mL

Add 611 uL to make enough for 6 slides, or up to 1 mL

Freezer 266A

antibody-mouse_igg_h_l_cross-adsorbed.pdf

Goat anti-mouse TRITC

Goat anti-mouse TRITC kdorfman Tue, 10/02/2018 - 19:23

Anti-Mouse IgG (whole molecule) TRITC conjugate

Sigma T 5393

26 mg protein/mL

minimum working dilution = 1:64

1 µL aliquots. Add up to 100 µL 1% BSA

Alexa Fluor 568 (A11004) (red) 2mg/mL

working concentration ~10µg/mL
dilute 1:200

aliquots are 110 µL 20µg/mL

add
110 µL 2% BSA in PBS
to make 220 µL 10µg/mL

Use 50 µL per coverslip (there is enough for 4 per tube)

Goat anti-rabbit FITC

Goat anti-rabbit FITC kdorfman Tue, 10/02/2018 - 20:42

Goat anti-rabbit IgG FITC

Sigma F0382

Dilute 1:80

f0382dat.pdf

Goat anti-rabbit red

Goat anti-rabbit red kdorfman Tue, 10/02/2018 - 19:25

Jackson Immuno 111-165-003

Cy3 Ex - 550 nm, em= 570 nm ("Red")

tubes : 2 µL. Add 398 µL PBS-Tw-Az to make 1:200

Use 50 µL per coverslip (there is enough for 4 per tube)

Goat anti-rat FITC

Goat anti-rat FITC kdorfman Tue, 10/02/2018 - 19:24

Sigma F6258

Each tube has 3 µL antibody

Add
192 µL PBS-Tw-Az
195 µL 2% BSA
=390 µL, enough for 7 reactions (50 µL/coverslip 37C 30min)

Goat anti-rat TRITC

Goat anti-rat TRITC kdorfman Tue, 10/02/2018 - 19:31

From Sigma AP136R EMB Millipore

ab peak = 550nm; emission peak = 570 nm

Bottle contains 2 mg. Add 1 mL H2O + 1 mL glycerol to make 1 mg/mL

aliquot 2 µL or 5 µL antibody, freeze

add 198 µL or 495 µL PBS-Tw-BSA

use 50 µL per coverslip, diluted to 1/100 (Try 37C 30 min)

Buffers

Buffers kdorfman Tue, 06/16/2009 - 17:51

Here are some commonly made buffers for biochemistry and molecular biology

HBSS

Sigma Buffer Reference Center

User Guide for GoldBio Buffers.pdf

Citric acid buffer

Citric acid buffer kdorfman Tue, 11/16/2021 - 20:03

Citric Acid – Na2HPO4 Buffer Preparation, pH 2.6–7.61

From Sigma Buffer Reference Center

Citric acid monohydrate, C6H8O7 • H2O, MW 210.14; 0.1 M contains 21.01 g/L.

Na2HPO4, MW 141.98; 0.2 M contains 28.40 g/L, or Na2HPO4 • 2H2O, MW 178.05; 0.2 M contains 35.61 g/L.

pH x mL 0.1 M-citric acid y mL 0.2-Na2HPO4
2.6 89.10 10.90
2.8 84.15 15.85
3.0 79.45 20.55
3.2 75.30 24.70
3.4 71.50 28.50
3.6 67.80 32.20
3.8 64.50 35.50
4.0 61.45 38.55
4.2 58.60 41.40
4.4 55.90 44.10
4.6 53.25 46.75
4.8 50.70 49.30
5.0 48.50 51.50
5.2 46.40 53.60
5.4 44.25 55.75
5.6 42.00 58.00
5.8 39.55 60.45
6.0 36.85 63.15
6.2 33.90 66.10
6.4 30.75 69.25
6.6 27.25 72.75
6.8 22.75 77.25
7.0 17.65 82.35
7.2 13.05 86.95
7.4 9.15 90.85
7.6 6.35 93.65

For 2021 QBoC

Need pH 3,4,5,6,7

Start with

  • Citric acid (100 mM) (borrowed from P-Chem)
    • 2.65 g
    • 125 mL H2O
  • Sodium phosphate dibasic (200 mM)
    • MW = 268.07
    • 200 mM = 8.042 g
    • 150 mL H2O

Then make

pH x mL 0.1 M-citric acid y mL 0.2-Na2HPO4
3.0 39.725 10.275
4.0 30.75 19.275
5.0 24.25 25.75
6.0 18.425 31.575
7.0 8.825 41.175

Test pH of 1:10, 1:2

  • pH remains constant when diluted to 20% with LB.
  • So make 50 mL of each diluted to 40% with LB. Students will dilute it 1:1 with LB
    • 20 mL buffer
    • 30 mL water
  • Filter steriize, aliquot for students

DEB

DEB kdorfman Mon, 12/19/2011 - 17:40

DNA Extraction Buffer for Gene & Genome

  • 100 mM NaCl
  • 50 mM Tris, pH8
  • 25 mM EDTA
  • 1% SDS

Make from stock solutions

To make mL DNA Extraction Buffer
add: mL 5M NaCl
add: mL 1M Tris, pH 8
add: mL EDTA 0.5M
add: mL 20% SDS
add: uL BMe AT THE LAST MINUTE!

Diluent B

Diluent B kdorfman Mon, 09/26/2022 - 20:21

Dilution buffer for NEB enzymes

Buffer composition:

Reagent Final concentration
NaCl 300 mM
Tris-HCl 10 mM
DTT 1 mM
EDTA 0.1 mM
BSA 500 ug/mL
glycerol 50%
pH 7.4

To make 50 mL for use, make 25 mL of 2x, then mix 1:1 with glycerol

25 mL 2x Diluent B

Reagent c1 c2 v1
NaCl 5M 0.3M 3 mL
Tris HCl 1M 0.01M 0.5 mL
DTT 1 M 1 mM 0.05 mL
EDTA 500 mM 0.1 mM 0.01 mL

plus BSA 25 mg

pH to 7.4, bring volume to 25, add glycerol to 50.

Filter sterilize

Diluent B info sheet from NEB

Edwards Extraction Buffer

Edwards Extraction Buffer kdorfman Tue, 03/22/2022 - 13:20

For quickie DNA extraction from Arabidopsis

  • 200 mM Tris pH 7.5
  • 250 mM NaCl
  • 25 mM EDTA
  • 0.5% SDS
To make mL DNA Extraction Buffer
add: mL 1M Tris, pH 7.5
add: mL 5M NaCl
add: mL EDTA 0.5M
add: mL 20% SDS

HBS (Ca-)

HBS (Ca-) kdorfman Tue, 12/27/2011 - 17:53

Calcium-free HBS

Make from dry ingredients:

To make mL Calcium-free HBS
start with g MgCl2
add: g KCl
add: g NaCl
add: g EGTA
add: g HEPES

Compound MW mM 1000 500 350 250 100 mL
MgCl2(6H2O) 203.3 0.493 0.1 0.05 0.035 0.025 0.01 g
KCl 74.56 2.67 0.2 0.1 0.07 0.05 0.005 g
NaCl 58.43 137.9 8 4 2.8 2 0.8 g
EGTA 380.4 0.1 0.038 0.0192 0.0134 0.0095 0.00384 g
HEPES 238.21 10 2.383 1.1915 0.858 0.596 0.238 g

raise pH to 7.3 with NaOH

1X HBS (ca-) from stock solutions

CHECK THIS

Ingredient stock M mM 0.25 0.5 1 2 L
MgCl.6H2O 1 0.49 0.0001 0.0002 0.0005 0.0010 mL
KCl 1 2.67 0.0007 0.0013 0.0027 0.0053 mL
NaCl 5 137.90 0.0069 0.0138 0.0276 0.0552 mL
HEPES 1 10 0. 025 0. 050 0. 100 0. 200 mL

pH 7.3 with NaOH

filter sterilize

HBS/Ca/Mg

HBS/Ca/Mg kdorfman Tue, 12/13/2011 - 20:32
Ingredient1 1X (mM) 10X (M)
CaCl2 0.9 0.009
MgCl2 0.493 0.00493
KCl 2.67 0.0267
NaCl 137.9 1.379
HEPES 10 0.1

  1. Concentrations from Lab 9, Bioimaging 2008, Dave Gross ↩︎

10X HBS (dry)

10X HBS (dry) kdorfman Fri, 08/09/2013 - 18:55

10X HBS/Ca/Mg from dry ingredients

To make mL 10X HBS/Ca/Mg
add: g CaCl2.2H20 (MW = 147.02)
add: g MgCl.6H2O (MW = 203.3)
add: g KCl (MW = 74.56)
add: g NaCl (MW = 58.43)
add: g HEPES (MW = 238.31)

pH 7.3 with NaOH

Filter sterilize

10X HBS (stocks)

10X HBS (stocks) kdorfman Fri, 08/09/2013 - 18:56

10 X HBS from stock solutions

To make mL 10X HBS/Ca/Mg
add: mL 1M CaCl2.
add: mL 1M MgCl
add: mL 1M KCl
add: mL 5M NaCl
add: mL 1M HEPES

pH 7.3 with NaOH

Filter sterilize

1X HBS (stocks)

1X HBS (stocks) kdorfman Fri, 08/09/2013 - 20:22
To make mL 10X HBS/Ca/Mg
add: mL 1M CaCl2.
add: mL 1M MgCl
add: mL 1M KCl
add: mL 5M NaCl
add: mL 1M HEPES

pH 7.3 with NaOH

Filter sterilize

HBSS

HBSS kdorfman Fri, 04/03/2020 - 14:57

Hank's Balanced Salt Solution

Gibco cat. #14025-092

stock bottle kept in tissue culture fridge

To make 1 Liter from dry ingredients

Components MW (mg/L) mM
Calcium Chloride (CaCl2) (dihyd.) 147 185 1.26
Magnesium Chloride (MgCl2-6H2O) 203 100 0.493
Magnesium Sulfate (MgSO4-7H2O) 246 100 0.407
Potassium Chloride (KCl) 75 400 5.33
Potassium Phosphate monobasic (KH2PO4) 136 60 0.441
Sodium Bicarbonate (NaHCO3) 84 350 4.17
Sodium Chloride (NaCl) 58 8000 137.93
Sodium Phosphate dibasic (Na2HPO4-6H20) 268 90.6 0.338
D-Glucose (Dextrose) 180 1000 5.56

Filter sterilize


To make from stock solutions:

Components Ci (M) cf (mM) 1000 500 250 mL
CaCl2 0.5 1.26 2.52 1.26 0.63 mL
MgCl2 1 0.493 0.493 0.247 0.108 mL
MgSO4 1 0.407 0.407 0.204 0.102 mL
KCl 1 5.33 5.33 2.67 0.131 mL
KH2PO4 1 0.441 0.441 0.22 0.11 mL
NaHCO3 0.5 4.17 8.34 4.17 0.209 mL
NaCl 5 137.93 27.59 13.79 6.9 mL
Na2HPO4 1 0.338 0.338 0.169 0.085 mL
Glucose - - 1 0.5 0.25 g

Filter sterilize

HEPES

HEPES kdorfman Tue, 12/20/2011 - 14:16

HEPES 1M

MW = 238.3

7.149 g / 30 mL

11.915 g / 50 mL

pH to 7.3 with NaOH pellets (~5 g/L)

Filter sterilize.

HotSHOT genomic DNA prep

HotSHOT genomic DNA prep kdorfman Tue, 06/21/2022 - 15:00

For fin clip, tail snip, or ear punch

  • Obtain tissue 2 mm
    • tail snip
    • ear punch
    • fin clip
  • Place tissue in 96 well plate
  • Add 75 uL Alkaline Lysis Reagent for mouse, 50 uL for fish
  • Heat to 95C 30 min (10 min to 1 hr)
  • Cool to 4C
  • Add 75 uL Neutralization Buffer for mouse, 50 uL for fish
  • Use 3 uL (1-5) pre PCR reaction

Notes:

  • DNA is suitable for PCR, not Southerns
  • Heating longer than 30 min does not increase [DNA]
  • pH of reagents does not need to be altered
  • DNA yield is similar for tail snips and ear punches
  • Too much tissue destroys PCR
  • Store DNA at 4C or 20C
HotSHOT DNA protocol handout

Alkaline Lysis Reagent

Alkaline Lysis Reagent kdorfman Tue, 06/21/2022 - 15:07

Alkaline Lysis Reagent for HotSHOT DNA prep

Recipe from Mike Charles 10/15/03; mikchar@umich.edu; retrieved from Craig Albertson's lab, recalculated for1 M NaOH - 10 M will damage your plastic tube!!

Reagent stock conc final conc amt
NaOH 1 M 25 mM 1.25 mL
EDTA 0.5 M 0.2 mM 20 uL
dI water to final volume 50 mL

Calculator

To make mL Alkaline Lysis Reagent
add: mL 1M NaOH
add: uL 0.5M EDTA
plus to final volume: mL H2O

pH =~12

Neutralization Buffer

Neutralization Buffer kdorfman Tue, 06/21/2022 - 15:09

Neutralization Buffer for HotSHOT DNA prep

Dilute stock Tris-HCl to 40 mM

pH =~5

PBS

PBS kdorfman Tue, 06/16/2009 - 17:52

Make 1X PBS from 10X

100 mL 10X + 900 mL H2O

10X PBS from Fisher

Fisher BP399-4 $51.07 as of 05/2020 ($34.45 January 2018!)

https://punchout.fishersci.com/shop/products/phosphate-buffered-saline-…

10X PBS from stock solutions

from Sigma ready-made 1X:

M ingredient
0.01 Na-K Phosphate
0.138 NaCl
0.0027 KCl
To make L 10X PBS
add: mL 5M NaCl
add: mL 1M KCl
add: mL 1M KH2PO4
add: mL 1M Na2HPO4.7 H2O (MW = 268.07)

pH to 7.3 with NaOH

sterilize

10X PBS from dry ingredients (Carrie's recipe)

To make L 10X PBS
add: g NaCl (MW = 58.44)
add: g KCl (MW = 74.55)
add: g KH2PO4 (MW = 136.09)
add: g Na2HPO4.7 H2O

pH to 7.3 with NaOH

sterilize

from stock solutions

from stock solutions kdorfman Wed, 11/13/2013 - 18:13

10X PBS from stock solutions

from Sigma ready-made 1X:

M (1X) M (10X) ingredient
0.01 0.10 Na-K Phosphate
0.138 1.38 NaCl
0.0027 0.027 KCl
calculated from Carrie's dry ingredients recipe:
To make L 10X PBS
add: mL 5M NaCl
add: mL 1M KCl
add: mL 1M KH2PO4
add: mL 1M Na2HPO4.7 H2O (MW = 268.07)

pH to 7.3 with NaOH

sterilize

OR

To match Sigma recipe:

Make 1M Na2HPO4 (base); 1 M KH2PO4 (acid)

Mix to pH 7.3

(should be ~38.25 mL Na2HPO4, 11.5 mL KH2PO4) from the Sigma Buffer Reference Center

pH mL Na Phos dibasic mL Na Phos monobasic
7.2 36.0 14.0
7.4 40.5 9.5

Then

To make L 10X PBS
add: mL 5M NaCl
add: mL 1M KCl
add: mL 1M K-Na Phosphate

PBX (for fish)

PBX (for fish) kdorfman Mon, 03/23/2020 - 13:57

0.5% Triton X in PBS

To make mL PBX
add: 10X PBS mL
and: 10% Triton-X mL

Plus water to final volume

PBX/BSA

PBX/BSA kdorfman Tue, 11/10/2020 - 18:44

PBX with 3% BSA for fish

To make mL PBX/BSA
add: g to PBX

PIPES

PIPES kdorfman Thu, 01/19/2012 - 16:43

0.5 M pH 6.7

(for transformation buffer for genetics)

MW (disodium salt) = 346.32

173.16 g/L

17.316 g/ 100 mL

Mix in ~80% of final volume

pH to 6.7 with 5M KOH

Filter sterilize

Aliquot

Freeze

Potassium phosphate

Potassium phosphate kdorfman Sat, 01/14/2012 - 04:58

1 M Potassium phosphate buffer

To make 100 mL of 1M potassium phosphate:

pH mL 1M K2HPO4 mL 1M KH2PO4
5.8 8.5 91.5
6.0 13.2 86.8
6.2 19.2 80.8
6.4 27.8 72.2
6.6 38.1 61.9
6.8 49.7 50.3
7.0 61.5 38.5
7.2 71.7 28.3
7.4 80.2 19.8
7.6 86.6 13.4
7.8 90.8 9.2
8.0 94.0 6.0

from http://ivaan.com/protocols/151.html

To make approximately: mL Potassium phosphate buffer
with: pH
start with approximately: mL 1M K2HPO4 (base)
and: mL 1M KH2PO4 (acid)

Check the pH with the meter.

Add more K2 to raise the pH, or K1 to lower it.

Dilute as necessary to achieve the desired concentration.

Sodium Phosphate

Sodium Phosphate kdorfman Mon, 12/12/2011 - 19:20

To make 100 mL 1M sodium phosphate at a given pH:

pH 1 M Na2HPO4 1 M NaH2PO4
8.0 93.2 ml 6.8 ml
7.8 89.6 ml 10.4 ml
7.6 84.5 ml 15.5 ml
7.4 77.4 ml 22.6 ml
7.2 68.4 ml 31.6 ml
7.0 57.7 ml 42.3 ml
6.8 46.3 ml 53.7 ml
6.6 35.2 ml 64.8 ml
6.4 25.5 ml 74.5 ml
6.2 17.8 ml 82.2 ml
6.0 12.0 ml 88.0 ml
5.8 7.9 ml 92.1 ml

Dilute to the desired concentration.

Sodium acetate buffer

Sodium acetate buffer kdorfman Tue, 11/16/2021 - 19:53

From Sigma Buffer Reference Center

Sodium Acetate – Acetic Acid Buffer PREPARATION | pH 3.7–5.61 Sodium acetate trihydrate, CH3COONa • 3H2O MW 136.09; 0.2 M contains 27.22 g/L.

x mL 0.2 M-NaOAc and y mL 0.2 M-HOAc mixed

pH @ 18 °C x mL 0.2 M-NaOAc y mL 0.2 M-HOAc
3.7 10.0 90.0
3.8 12.0 88.0
4.0 18.0 82.0
4.2 26.5 73.5
4.4 37.0 63.0
4.6 49.0 51.0
4.8 59.0 41.0
5.0 70.0 30.0
5.2 79.0 21.0
5.4 86.0 14.0
5.6 91.0 9.0

Sodium borate buffer

Sodium borate buffer kdorfman Fri, 11/05/2021 - 14:10

(to neutralize acid wash during anti-5mC staining)

1L 1M

g ingredient
61.83 g Boric acid (H3BO3)
10 g NaOH

water to 1L

100 mL 100mM

g ingredient
0.6183 g Boric acid (H3BO3)
0.1 g NaOH

water to 100 mL

T10E1

T10E1 kdorfman Mon, 12/19/2011 - 17:54

Tris 10 mM, EDTA 1 mM, pH 8

Make from stock solutions

To make mL T10E1
add: mL 1M Tris pH 8
add: mL 0.5M EDTA

ingredient cf ci 25 50 mL
Tris pH 8 10 mM 1000 mM 0.25 0.5 mL
EDTA 1 mM 500 mM 0.05 0.1 mL

T10E5

T10E5 kdorfman Mon, 12/19/2011 - 18:25

Tris 10 mM, EDTA 5 mM, pH 8

Make from stock solutions

To make mL T10E5
add: mL 1M Tris pH 8
add: mL 0.5M EDTA

ingredient cf ci 25 50 mL
Tris pH 8 10 mM 1000 mM 0.25 0.5 mL
EDTA 5 mM 500 mM 0.25 0.5 mL

TAE

TAE kdorfman Tue, 06/16/2009 - 17:52

Tris-Acetate-EDTA buffer for agarose gel electrophoresis

1X TAE

1X TAE kdorfman Tue, 06/16/2009 - 19:02
To make L 1X TAE
add: mL 50X TAE

plus water to final volume

50X TAE stock

50X TAE stock kdorfman Tue, 06/16/2009 - 19:01

Buy from Fisher TAE 50x, pH 8.3, 4L, part #BP13324

from Maniotis

To make mL 50X TAE
add: g Tris base
add: mL glacial acetic acid
add: mL 0.5M EDTA
OR add: g EDTA

pH to 8 with acetic acid or NaOH

should this be 8.4?

Ingredient cf (mM) 1000 750 600 500 mL
Tris 2000 242 181.5 145.2 121 g
Acetic acid 1000 57.1 42.9 34.3 28.6 mL
EDTA 0.5 M 50 100 75 60 50 mL
OR
EDTA 50 18.62 13.965 11.172 9.31 g

pH to 8 with acetic acid or NaOH

Maniotis says to autoclave, but the salt concentration is so high that nothing will grow in it if you don't.

Taq dilution buffer

Taq dilution buffer kdorfman Fri, 05/31/2013 - 17:21

To dilute Takara Ex-Taq

Store in freezer. Won’t freeze solid. Should keep forever.

To make mL Taq dilution buffer
add: mL Tris 1M pH 8
add: mL KCl 1M
add: mL EDTA 0.5M
add: mL DTT 1M
add: mL Tween 20
add mL Nonidet P-40
add mL glycerol

bring to final volume, filter sterilize in BSC, freeze

Ex-Taq user manual

Culture Media

Culture Media kdorfman Fri, 04/27/2018 - 15:16

Bacterial media

Bacterial media kdorfman Thu, 01/17/2013 - 18:53

Min med + HMMLT

Freezing bacteria

Freezing bacteria kdorfman Sat, 12/05/2015 - 18:44

To Freeze:

  • Pick a new colony, grow overnight in LB

  • inoculate LB from the overnight culture

  • Put 0.15 mL glycerol into sterile cryovial.

  • Add 0.85 mL mid-log culture (OD =~0.4). Pipette up and down to mix thoroughly

  • Freeze. Store in -80 if possible.

To thaw:

  • Scrape surface of frozen stock with sterile stick

  • Streak on agar plate

LB Broth

LB Broth kdorfman Fri, 09/25/2020 - 17:54

LB broth from mix

LB broth from mix kdorfman Tue, 05/28/2013 - 17:48

LB Broth Miller Luria-Bertani

Fisher DF0446-17-3 500 g $40.61.

25 g/L

To make mL LB broth
add: grams LB mix, stir till dissolved.
autoclave: minutes

LB from scratch

LB from scratch kdorfman Fri, 09/25/2020 - 17:38

LB (Miller) from scratch

To make mL LB broth from scratch
add: grams tryptone
add: grams yeast extract
add: grams NaCl
autoclave: minutes

LB agar

LB agar kdorfman Thu, 01/17/2013 - 18:55

LB agar granules

LB agar granules kdorfman Tue, 06/25/2019 - 20:01

Make with granulated LB (Miller) agar, Fisher BP9724

To make LB agar plates
at mm plate diameter
pour mL per plate
start with: mL water
addg LB agar *granules*, stir till dissolved
autoclave for: minutes

LB agar powder

LB agar powder kdorfman Tue, 06/25/2019 - 20:00

Make with LB (Miller) Agar powder, Difco 244510

To make LB agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add g LB agar, stir till dissolved
autoclave for: minutes

LB broth + agar

LB broth + agar kdorfman Tue, 06/25/2019 - 20:00

25 g LB + 15 g agar /liter

To make LB agar plates (100 mm)
you'll need: mL water
add: g LB broth, stir till dissolved
add: g agar, leave stir bar in
autoclave: minutes

LB antibiotic

LB antibiotic kdorfman Wed, 10/01/2014 - 16:34

Table of antibiotic stability in poured agar

To make:
mL LB
with:
µg/mL antibiotic
and the antibiotic stock is:
mg/mL. Cool slightly.
then add:
µL antibiotic stock solution

line antibiotic
black Carbenicillin (ampicillin)
green IPTG

LB-sugars

LB-sugars kdorfman Wed, 10/01/2014 - 16:19

LB + sugars for Lac-operon work

sugar (or analog) MW
mono saccharide 180.2
disaccharide 342.3
IPTG 238.3

glucose, galactose, or fructose
To make mL LB broth
that is: M sugar,
add: g monosaccharide (glucose, galactose, or fructose)

lactose, maltose, sucrose
To make mL LB broth
that is: M sugar,
add: g disaccharide (lactose, maltose, sucrose)

Lactose is not very soluble in cold water.. Boil water, then set it to stir on a stir plate. Gradually tap in the lactose powder.

IPTG
To make mL LB broth
that is: mM IPTG,
add: µL 1M IPTG

Filter sterilize

Add 50 µg/mL Kanamycin as indicated here.

MacConkey agar

MacConkey agar kdorfman Thu, 01/17/2013 - 19:41

MacConkey Agar

  • Fisher 212122 2 kg (Difco)
  • Krackeler 10-211387 500g (via Sigma)
  • HiMedia MacConkey agar, granulated GM081 (Fisher NC1876763, VWR 95020-204)

to distinguish Lac+ and Lac- bacterial strains.

50 g/L

(If you are going to make more than one bottle, turn on 65C waterbath in tissue culture room)

Turn on the stir-plate heat to 105C.

Withhold 50 mL of water to rinse the mouth of the bottle.

Heat the rest of the water to boiling in the bottle in the microwave.

Measure out the agar mix while the water is heating

Add the stir bar (CAREFULLY!) to the bottle of hot water

Put the bottle on the stir-plate

Add the agar mix to the bottle

Use the reserved water to rinse the granules stuck in the neck down to the liquid.

Stir and heat until granules are completely dissolved.

Cap loosely, wrap with foil, and autoclave.

(If you make more than one bottle, the extras go in the water bath till you are ready to pour)

To make Maconkey agar plates
at mm plate diameter
pour mL per plate
start with: mL hot water
add g Maconkey agar, heat to boiling, stir till dissolved
autoclave for: minutes

leave stir bar in

put bottles on stir plate near sterile hood until handle-able

If you accidentally buy

MacConkey + bile+ CV

MacConkey + bile+ CV kdorfman Mon, 01/08/2024 - 16:00

If you accidentally buy MacConkey without

  • bile salts (1.5 g/L)
  • Crystal violet (0.001g/L)
  • NaCl (5 g/L)
To make Maconkey agar plates
at mm plate diameter
pour mL per plate
start with: mL hot water
add g Maconkey agar (without bile, CV, NaCl)
add g bile salts
add g NaCl
add mL 1% crystal violet solution, heat to boiling, stir till dissolved
autoclave for: minutes

Min med for GFP cells

Min med for GFP cells kdorfman Tue, 10/23/2018 - 15:53

From https://wahoo.cns.umass.edu/node/857

For SfGFP cells, try M9 + 0.2% glycerol + 0.01 mM FeSO4

To make mL min med
start with mL 10X M9
add: mL 100% glycerol
add: mL 10mM FeSO4
Bring to final volume, filter sterilize

Mueller Hinton agar

Mueller Hinton agar kdorfman Thu, 01/17/2013 - 20:05

90922 Mueller Hinton Broth 2 from Sigma

22 grams per liter, consisting of:

Casein acid hydrolysate 17.5
Beef extract 3.0
Starch 1.5

Final pH 7.3 +/- 0.2 at 25°C

To make Mueller-Hinton plates (100 mm)
you'll need: mL water
add: g MH broth, stir till dissolved
add: g agar, leave stir bar in
autoclave: minutes

P-Glo Bacterial media

P-Glo Bacterial media kdorfman Mon, 08/26/2019 - 22:26

Make 60 mm plates

2019 - for QBoC:

  • transformed pglo plasmid (1660405EDU) from the BioRad kit into
  • DH5 alpha E. coli (Invitrogen 18265-017) from the -80
  • Lyophilized cells (BioRad 1660408EDU) did not arrive in time

Used the Invitrogen protocol.

Plated on:

  • LB
  • LB amp
  • LB amp ara

Saw gfp colonies on the arabinose plates. Collected, grew up in LB amp, froze in 15% glycerol.

Bacterial Transformation Lab Protocol.pdf
p-glo instructor manual
DH5alpha subcloning protocol

LB-Amp-Ara

LB-Amp-Ara kdorfman Mon, 08/26/2019 - 22:27

LB with Ampicilin or Carbenicilin and arabinose

Arabinose stock 200mg/mL in water, sterilized by filtration (=100 X)

Final concentration = 2 mg/mL

  • Arabinose MW = 150.13
  • 2 mg/mL = 2 g/L x 1 mol/150.13 g = 0.013M = 13 mM

Carbenicilin stock is 1000X

minimal medium (glycerol)

minimal medium (glycerol) bcrcstaff Wed, 11/14/2018 - 17:34

For motility strains

To make mL min med
start with mL 10X M9
add: mL 200X HMLTT
add: mL 100% glycerol
add: mL H2O
add: mL 1M MgSO4
add: uL 1M CaCl2, stir if it precipitates
Bring to final volume

Cell culture media

Cell culture media kdorfman Tue, 10/11/2011 - 19:24

FBS aliquots

FBS aliquots kdorfman Tue, 10/11/2011 - 20:15

FBS comes in 500 mL bottles.

Aliquot:

volume (mL) # aliquots for
50 3 500 mL DMEM or non-CO2 medium
37.5 4 500 mL F10-Ham's
25 4 250 mL DMEM or non-CO2 medium
18.75 4 250 mL F10 Ham's
6.25 4 to make freezing media

2013: Try

  • Fisherbrand™ Research Grade Fetal Bovine Serum
  • 03-600-511
  • 500 mL for $133

Krackeler 45-F0926-500ML $145

Carrie's brand: Atlanta Biologicals Premium S11150 ~$300

Also use Krackeler 12103C ~$314

2019 Genessee GenClone FetalPURE™ Cat #: 25-525 $247.45

Antibiotic/Antimycotic

Antibiotic/Antimycotic kdorfman Tue, 10/11/2011 - 20:21

Anti-Anti comes in 100 mL bottles.

Aliquot:

volume (mL) # aliquots for
10 1 1 L
5 12 500 mL
2.5 12 250 mL

Keep frozen until ready to use.

Fisher SV30079.01, $20

DC5 (DMEM for canine cancer)

DC5 (DMEM for canine cancer) kdorfman Thu, 08/17/2023 - 20:00

from Kathleen Arcaro

To make mL D5 DMEM with pyruvate, glucose, glutamine
start with mL water
add: g DMEM (Gibco 12800-058/ fisher 12800017)
add: g NaHCO3 if there is none already in the DMEM
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL insulin solution 10 mg/mL in HEPES
add: mL amino acid solution
add: mL cosmic calf serum
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

insulin solution 10 mg/mL in HEPES

amino acid solution

DMEM for Canine Cancer Cells

DMEM

DMEM kdorfman Tue, 10/11/2011 - 19:30

Medium for 3t3 fibroblasts and B16 cells

To make mL DMEM
start with mL water
add: g DMEM*
add: g NaHCO3
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL FBS
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

  • if using DMEM without pyruvate, add 1 mL Na Pyruvate (100mM) per 100 mL medium
Ingredient supplier cat # 1 L 500 mL 250 mL
DMEM 13.4 6.7 3.35 g
NaHCO3 3.7 1.85 0.925 g
HEPES 1.3 0.65 0.325 g
FBS Krackeler 12103C 100 50 25 mL
anti/anti Fisher SV30079.01 10 5 2.5 mL

Mix DMEM, NaHCO3, HEPES in about 70% of the final volume of dH2O.
Initial pH ~7.5
Adjust pH to 7.2 with HCl (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.

F10-Ham's

F10-Ham's kdorfman Tue, 10/11/2011 - 19:49

for all LLCPk cell lines

Thaw FBS and anti-anti first!

To make mL F10-Ham's
start with mL water
add: g F10 (Hams) (Sigma N6635)
add: g Optimem (Invitrogen 226000-050)
add: g NaHCO3
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL FBS
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

Ingredient supplier cat # 1 L 500 mL 250 mL
F10 (Ham's) Sigma N6635 4.9 2.45 1.225 g
Optimem Invitrogen 22600-050 6.8 3.4 1.7 g
NaHCO3 1.8 0.9 0.45 g
HEPES 0.66 0.33 0.165 g
FBS Krackeler 12103C 75 37.5 18.75 mL
anti/anti Fisher SV30079.01 10 5 2.5 mL

Mix F10, Optimem, NaHCO3, HEPES in about 70% of the final volume of dH2O.

Initial pH ~7.1
Adjust pH to 7.2 (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Refrigerate

For serum free, replace serum with distilled water

Freezing media

Freezing media kdorfman Tue, 10/11/2011 - 20:42

Medium with 15% DMSO and 20% serum to protect cells in liquid nitrogen.

F10 Hams for freezing (initial serum concentration = 0.075)

To make mL F10-Ham's for freezing
add: mL F-10 Ham's
add: mL serum
add: mL DMSO

DMEM for freezing (initial serum concentration = 0.1)

To make mL DMEM for freezing
add: mL DMEM
add: mL serum
add: mL DMSO

Filter sterilize

McCoy's 5A

McCoy's 5A kdorfman Tue, 09/19/2023 - 15:19

McCoy's 5A (modified) (Fisher 16600-082)

For HCT116 cells (a human colorectal carcinoma cell line initiated from an adult male. The cells are adherent with an epithelial morphology. Following implantation into immunocompromised mice, the cells form primary tumors and distant metastases. In vitro, HCT116 cells grow with a doubling time of about 18 hours.)

Drew's recipe:

10% FBS, 1% Pen-strep

  • 500 mL McCoy
  • 55 mL FBS
  • 5.5 mL pen-strep

OR:

  • remove 55 mL from the 500 mL McCoy's from Fisher
  • add 50 mL FBS
  • add 5 mL pen strep (e.g., MP 1670246)
  • resterilize

Filter sterilize

If you havemL McCoy's
mix withmL FBS
andmL Pen-Strep
for a final volume ofmL McCoy's ready to use

Non-CO2 Media

Non-CO2 Media kdorfman Tue, 12/27/2011 - 18:37

Try this: http://www.thermofisher.com/us/en/home/life-science/cell-culture/mammal…

FluoroBrite™ DMEM Media

serum-free

serum-free kdorfman Wed, 10/19/2011 - 15:07

Non-CO2 serum-free medium

For working with live cells at the microscope

To make mL non CO2 medium
start with mL water
add: g MEM (Sigma M3024-1L)
add: g HEPES, then pH to 7.3 (initial pH = ~6.3)
add: mL Na pyruvate 100 mM (thermo SH30239.01)
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

Ingredient supplier cat # 1000 500 250 mL
MEM Sigma M3024-1L 13.4 6.7 3.35 g
HEPES Acros 172571000 1.3 0.65 0.325 g
Na pyruvate 100 mM Thermo SH30239.01 10 5 2.5 mL
anti/anti Fisher SV30079.01 10 5 2.5 mL

Mix MEM and HEPES in about 70% of the final volume of dH2O.

Initial pH ~6.3
Adjust pH to 7.3
Add appropriate aliquot of antibiotic/antimycotic, Na pyruvate
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.

with serum

with serum kdorfman Tue, 10/11/2011 - 20:00

Non-CO2 Medium

For working with live cells at the microscope, when serum is needed for normal division.

To make mL F10-Ham's
start with mL water
add: g MEM (Sigma M3024-1L)
add: g HEPES, then pH to 7.3 (initial pH = ~6.3)
add: mL Na pyruvate 100 mM (thermo SH30239.01)
add: mL FBS
add: mL anti/anti

bring to final volume, filter sterilize in BSC, refrigerate

Ingredient supplier cat # 1 L 500 mL 250 mL
MEM 13.4 6.7 3.35 g
HEPES 1.3 0.65 0.325 g
Na pyruvate 100 mM 10 5 2.5 mL
FBS Krackeler 12103C 100 50 25 mL
anti/anti Fisher SV30079.01 10 5 2.5 mL

Mix MEM and HEPES in about 70% of the final volume of dH2O.

Initial pH ~6.3
Adjust pH to 7.3
Add appropriate aliquot of FBS, antibiotic/antimycotic, Na pyruvate
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.

Trypsin

Trypsin kdorfman Tue, 01/19/2016 - 18:58

Try Fisher 12-605-010

Stable at room temp!

Gibco™ TrypLE Express Enzyme (1X), Phenol Red

Animal origin-free, recombinant enzyme

$20.10 -for 100 mL

E2 medium (Zebrafish)

E2 medium (Zebrafish) kdorfman Sun, 01/13/2019 - 21:48

Use the attached file while this set of pages is under construction

E2 Embryo Medium final Working Solution: 0.5X E2

To make 20 L, start with 19 L RO water, and add

ml component
100 100X E2A
20 500X E2B
20 500X E2C
10 0.1% MeBlue (optional)

Then bring to final volume

E2_solution.pdf

0.1% Methylene Blue

0.1% Methylene Blue kdorfman Thu, 03/19/2020 - 14:53

1 gram of methylene blue per liter of RO water.

Shake well to dissolve.

Store at room temp.

0.5X E2A

0.5X E2A kdorfman Thu, 03/19/2020 - 14:54

E2A (100x)

E2A (100x) kdorfman Thu, 03/19/2020 - 14:51

FOLLOW THE RECIPE IN THE ATTACHED FILE (omitting methylene blue). THIS PAGE IS NOT DONE YET

100X E2:

1.5 M NaCl

50 mM KCl

100 mM MgSO4

15 mM KH2PO4

5 mM Na2HPO4

To make mL E2 medium
add: mL 5 M NaCl
add: mL 1 M KCl
add: mL 1 M MgSO4
add: mL 1 M KH2PO4
add: mL 1M Na2HPO4

E2A Buffer Mix

E2A Buffer Mix kdorfman Thu, 03/19/2020 - 14:52

E2B 500X

E2B 500X kdorfman Thu, 03/19/2020 - 14:52

E2C 500X

E2C 500X kdorfman Thu, 03/19/2020 - 14:53

Naegleria medium

Naegleria medium kdorfman Wed, 01/17/2018 - 14:34

Medium 21

Medium 21 kdorfman Wed, 12/15/2021 - 21:44

Mark with 2 red lines

Add 1 black line for carbenicillin (ampicillin)

Add 1 green line for IPTG

M21 from stocks

M21 from stocks kdorfman Wed, 12/15/2021 - 21:19
To make plates (100 mm)
(for a final volume of mL of medium)
start with: mL water
add: mL 1M K2HPO4
add: mL 1M KH2PO4
add: g Difco Bacto Peptone, and bring to final volume
add: g agar
autoclave for: minutes

Add 50 - 100 µg/mL ampicillin (carbenicillin) and 1 mM IPTG if needed

Medium 21 from dry

Medium 21 from dry kdorfman Tue, 12/14/2021 - 21:09
To make plates (100 mm)
you'll need: mL water
add: g K2HPO4
add: g KH2PO4
add: g peptone (Difco Bacto Peptone, not proteose)
add: g agar
autoclave for: minutes

If required, add IPTG 0.5mM (0.5 mL 1M per L medium) to 1 mM (1 mL 1M per L medium),

and ampicillin

NM from dry

NM from dry kdorfman Wed, 01/17/2018 - 14:34
To make plates (100 mm)
you'll need: mL water
add: g K2HPO4
add: g KH2PO4
add: g peptone (Difco Bacto Peptone, not proteose)
add: g agar
add: g dextrose
autoclave for: minutes

If required, add IPTG 0.5mM (0.5 mL 1M per L medium) to 1 mM (1 mL 1M per L medium),

and ampicillin

NM from stock solutions

NM from stock solutions kdorfman Wed, 01/17/2018 - 14:35

DIW (pure deionized water) 780 ml

1M K2HPO4 (dibasic) 4 mL

1M KH2PO4 (monobasic) 16 mL

Difco Bacto Peptone (not Proteose Peptone) 3.2 g

Difco Bacto Agar 12.0 g

Mix by swirling (no need to dissolve). Autoclave 10 min. Cool to 45–50° in a water bath, and pour plates about ½ full.

To make plates (100 mm)
(for a final volume of mL of medium)
start with: mL water
add: mL 1M K2HPO4
add: mL 1M KH2PO4
add: g peptone (Difco Bacto Peptone, not proteose)
add: g agar
autoclave for: minutes

Plant growth media

Plant growth media kdorfman Thu, 05/23/2013 - 16:02

10 g agar per L

germination_media.xlsx

1/2 MS low Iron

1/2 MS low Iron kdorfman Tue, 02/15/2022 - 15:00

1/2 Murashige & Skoog, 1µM iron medium

MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM, 1/2 MS is 5µM

FeSO4 stock is 10 mM, which is 10,000x 1 µM

To make plates low-iron 1/2 MS
start with mL ddH2O (~ 60% final volume)
add: mL 10X macronutrients
add: mL boric acid 1000X
add: mL cobalt chloride 10,000X
add: mL cupric sulfate 10,000X
add: mL ferrous sulfate 10 mM
add: mL KI 10,000X
add: mL manganese sulfate 1000X
add: mL moybdic acid 10,000X
add: mL zinc sulfate 1000X
add: g MES, pH to 5.7 with KOH
bring to mL final volume
add: g bacto- or phyto-agar
autoclave for minutes

Fern Culture Media

Fern Culture Media kdorfman Thu, 01/11/2024 - 19:16

C Fern Manual

Carolina C-Fern materials

Pre-made fern medium, Carolina 156780, sold in 60 mL or 400 mL

Carolina recommends 10 mL per plate. I use 9 mL

To get gametophytes in all stages, sow fern spores in 12 60 mm dishes 4 times:

  • 1/15
  • 1/22
  • 1/29
  • 2/2 or Friday 2/3
C-Fern Manual

Fern Medium from scratch

Fern Medium from scratch kdorfman Fri, 01/12/2024 - 21:18

To make one liter
800 mL water
10X macronutrients: 100mL
200X micronutrients: 5 mL
100X Chelated Iron solution: 10 mL

pH to 6 with NaOH

Bring final volume to 1 L

Add 10 g Bacto agar (others may not work)

Autoclave 15 min

10X fern macronutrients

10X fern macronutrients kdorfman Fri, 01/12/2024 - 21:24

200X fern micronutrients

200X fern micronutrients kdorfman Fri, 01/12/2024 - 21:26

Fern medium from powder

Fern medium from powder kdorfman Wed, 01/24/2024 - 14:20

Carolina 156782

Mix contents of vial with 800 mL water

pH to 6 (NaOH)

Bring to 1 L

For single batch:

  • Add 10g Bacto Agar

  • Autoclave 45 min

For small batches:

  • Aliquot 167 mL into 6 200 mL media bottles

  • Add 1.67 g Bacto Agar to each bottle

  • Autoclave 30 min

LPGM

LPGM kdorfman Wed, 08/31/2016 - 20:24

Lilly pollen growth medium

205 mM (7%) sucrose (See Sugars.)

1.6 mM H3BO3

0.1 mM CaCl2

15 mM MES

To make mL LPGM
at % sucrose
add: mL 150 mM MES
add: uL 10 mM CaCl2
add: uL 160 mM Borate, pH to 5.7 with KOH
add: g sucrose

Either make fresh each day, or filter sterilize for all the labs.

Stock solutions:

150 mM MES

10 mM CaCl2

160 mM Borate

MS high salt

MS high salt kdorfman Mon, 02/03/2014 - 15:33

150 too high - try 100mM for F 2015

150 mM NaCl

10 g agar/L

Make regular MS, then add 0.03 mL 5M NaCl per mL medium

100 mM NaCl

0.02 mL 5M NaCl per mL medium

MS iron-free

MS iron-free kdorfman Thu, 05/23/2013 - 17:46

Murashige & Skoog 1µM iron medium

MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM

FeSO4 stock is 10 mM, which is 10,000x 1 µM

10 g agar/L

To make plates low-iron MS
start with mL ddH2O (~ 60% final volume)
add: mL 10X macronutrients
add: mL boric acid 1000X
add: mL cobalt chloride 10,000X
add: mL cupric sulfate 10,000X
add: mL KI 10,000X
add: mL manganese sulfate 1000X
add: mL moybdic acid 10,000X
add: mL zinc sulfate 1000X
add: g MES, pH to 5.7 with KOH
bring to mL final volume
add: g bacto- or phyto-agar
autoclave for minutes

MS low iron

MS low iron kdorfman Thu, 05/23/2013 - 17:40

Murashige & Skoog 1µM iron medium

MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM

FeSO4 stock is 10 mM, which is 10,000x 1 µM

To make plates low-iron MS
start with mL ddH2O (~ 60% final volume)
add: mL 10X macronutrients
add: mL boric acid 1000X
add: mL cobalt chloride 10,000X
add: mL cupric sulfate 10,000X
add: mL ferrous sulfate 10 mM
add: mL KI 10,000X
add: mL manganese sulfate 1000X
add: mL moybdic acid 10,000X
add: mL zinc sulfate 1000X
add: g MES, pH to 5.7 with KOH
bring to mL final volume
add: g bacto- or phyto-agar
autoclave for minutes

MS medium

MS medium kdorfman Thu, 05/23/2013 - 16:02

1/2 MS

1/2 MS kdorfman Wed, 12/22/2021 - 20:25
To make 30 mL plates
start with: mL water initial volume*
add: mL 10X macronutrients [1]
add: mL 10X micronutrients [2]
add: g MES. pH to 5.7 w/ 1M KOH**
bring volume to: mL water final volume
add: g bacto or phyto agar
autoclave for: minutes

*Add the other salt mixtures to the water to prevent precipitation

**pH to 5.7 with 1M KOH (initial pH = ~3.66) (needs~21 drops or ~720 µL/L)

Autoclave with stir bar in flask or bottle

Stir until cool enough to handle

Pour 30 mL per plate (use the deep ones)

Pour 50 mL per square plate (20 plates per liter)

[1]: Sigma M 0654 Murashige and Skoog basal salt macronutrient solution (Krackeler 45-M0654-1L-EA) ~$26

[2]: Sigma M 0529 Murashige and Skoog basal salt micronutrient solution (Krackeler 45-M0529-1L-EA) ~$26

1X MS

1X MS kdorfman Wed, 12/22/2021 - 20:37
To make 30 mL plates
add: mL water initial volume*
add: mL 10X macronutrients [1]
add: mL 10X micronutrients [2]
add: g MES. pH to 5.7 w/ 1M KOH**
bring volume to: mL water final volume
add: g bacto or phyto agar
autoclave for: minutes

*Add the other salt mixtures to the water to prevent precipitation

**pH to 5.7 with 1M KOH (initial pH = ~3.66) (needs~21 drops or ~720 µL/L)

Autoclave with stir bar in flask or bottle

Stir until cool enough to handle

Pour 30 mL per plate (use the deep ones)

[1]: Sigma M 0654 Murashige and Skoog basal salt macronutrient solution (Krackeler 45-M0654-1L-EA) ~$26

[2]: Sigma M 0529 Murashige and Skoog basal salt micronutrient solution (Krackeler 45-M0529-1L-EA) ~$26

Slime mold media

Slime mold media kdorfman Fri, 04/14/2023 - 18:18

Saburaud dextrose agar

Saburaud dextrose agar kdorfman Fri, 04/14/2023 - 18:19

Wikipedia entry

Before you start:

  • check for 20% glucose, filter sterilized; make more if needed
  • warm the glucose (a ~60C oven is best; use a 37 incubator if necessary)
  • sterilize a graduated cylinder if volume of glucose to be added is more than 50 mL

per L:

  • 40 g glucose
  • 10 g peptone
  • 20 g agar
  • pH 5.6
To make mL Saburaud
start with: mL water
add: grams peptone
adjust pH to 5.6
bring volume tomL water
add: grams agar
autoclave for: minutes
aseptically add: mL sterile 20% glucose

VTM

VTM kdorfman Fri, 04/03/2020 - 14:42

Viral Transport Medium

Equipment and materials needed

  • Sterile Hood
  • Waterbath at 56C to heat-inactivate FBS
  • Sterile serological pipets
  • Sterile 15 mL tubes
  • Filter sterilization

Reagents

Viral Transport Medium (CDC)

Worm media

Worm media kdorfman Fri, 04/01/2016 - 16:28

Freezing medium

Freezing medium kdorfman Fri, 04/01/2016 - 16:30

Per 1 L

  • 100 mL 10X M9 salts
  • 240 mL glycerol
  • 300 µL 1M MgSO4
  • water to 1L

Filter sterilize

M9 for worms

M9 for worms kdorfman Fri, 04/01/2016 - 18:11

Per 1 L:

  • 100 mL 10X M salts
  • 300 µL 1M MgSO4
  • water to 1 L

Filter sterilize

NGM

NGM kdorfman Fri, 04/01/2016 - 16:31

Per Liter of medium (~75 plates):

  • 975 mL Water
  • 3 g NaCl
  • 2.5 g Peptone (Fisher BP1420-500 $78.80)
  • 17 g Bactoagar

Autoclave with stir bar inside

Cool to 55C in a 55C water bath

Add per L (see recipes in stock solutions):

  • 1 mL cholesterol (5 mg/mL in 95% EtOH)
  • 1 mL CaCl2 (1 M, STERILE)
  • 1 mL MgSO4 (1 M, STERILE)
  • 25 mL K-phosphate buffer (1M, pH 6.0, STERILE1)

Swirl flask to mix

Dispense 10 mL into each 60mm dish.

Stack 10 high

Let stand for ~48 hours for condensation to evaporate

Pack in sterilized plastic boxes.

To make NGM agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g peptone, stir till dissolved
addg NaCL, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add: mL 5mg/mL cholesterol
add: mL sterile 1M CaCl2
add: mL sterile 5mg/mL 1M MgSO4
add: mL sterile K-phosphate buffer , pH 6

put bottles on stir plate near sterile hood until handle-able

  1. 3.3 mL K2HPO4 + 21.7 mL KH2PO4 ↩︎

Yeast media

Yeast media kdorfman Mon, 02/08/2016 - 15:03
284 Yeast Plate Code.docx

284 Yeast Plate Code

284 Yeast Plate Code kdorfman Wed, 08/23/2023 - 15:49
Medium color code tape color
MV 1 blue line orange
MV-Ade 2 blue lines red
YEAD 1 black, 1 blue teal
YED 1 red line pink
YEKAc 1 black line green (use up the orange ones )
YEPAD 1 black, 1 red, 1 blue white
Yeast Plate Codes

MV + Ade

MV + Ade kdorfman Wed, 10/18/2017 - 16:42

For Bio 284

  • 0.15 g YNB
  • 0.52 g ammonium sulfate
  • 2.0 g agar
  • 82 mL H2O
  • 8.0 mL 1 mg/mL adenine
  • 10.0 mL 20% glucose (final conc = 2g/100 mL)

Minimal Vitamin Medium plus Adenine

Label plates with "+ ADE"

Make sure it's a plus sign!

Autoclave an appropriately sized graduated cylinder to measure the glucose and adenine solutions.

To make MV+ADE agar plates
pour mm plate diameter
pour mL per plate
start with: mL water
add: g YNB*, stir till dissolved
add: g ammonium sulfate, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
add asceptically: mL filter-sterilized adenine (1mg/mL)
for a final volume of mL

Mark with two blue lines

*Yeast Nitrogen Base without amino acids and ammonium sulfate

MV

MV kdorfman Mon, 02/08/2016 - 15:04

Minimal Vitamin Medium for Bio 284

Autoclave an appropriately sized graduated cylinder to measure the glucose solution.

To make MV agar plates
pour mm plate diameter
pour mL per plate
start with: mL water
add: g YNB*, stir till dissolved
add: g ammonium sulfate, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
for a final volume of mL

*Difco Yeast Nitrogen Base w/o Amino Acids and w/o Ammonium Sulfate

Mark with a single blue line

SC -Leu High Ade

SC -Leu High Ade kdorfman Sun, 01/19/2020 - 16:39

Plates for Jeff Laney's Cell & Molecular Biology Lab

8X SC-HLT hi Ade concentrate

8X SC-HLT hi Ade concentrate kdorfman Fri, 01/22/2021 - 22:01

Concentrate to make SC-HLT High Ade agar

add 125 mL heated concentrate to autoclaved 875 mL water + 20 g agar right out of the autoclave and still hot

to make enough concentrate for:
Liter(s) SC-HLT high Ade agar medium
start with
mL water
add:
g YNB
and:
g ammonium sulfate
and:
g SC -HLT
autoclave for:
minutes (to get it into solution)
while it's still hot, add:
g glucose
and:
g Adenine hemi sulfate
and:
g tryptophane
and:
mL his 100X concentrate.
Bring to a final volume of:
mL 8X SC-L high Ade and filter sterilize

Laney's SC high ade

Laney's SC high ade kdorfman Sun, 01/26/2020 - 16:22

From Jeff Laney's recipe:

Per liter of medium, mix the following asceptically, and pre heat:

Component Volume (mL)
10X SC-AHLT 100
10X YNB+AS 100
10X 20% glucose 100
10X Adenine hemi sulfate 100
50X Tryptophan 20
100X Histidine HCl 10

Add to 20g agar in 667.5 mL water, autoclaved 30 minutes

Mark with one green line.

SC -Leu High Ade 4x + powder

SC -Leu High Ade 4x + powder kdorfman Sun, 01/26/2020 - 16:26

SC -Leu High Ade 4x + powder

Use the 4x SC-AHLT to mix dry ingredients

(4x already made; not enough dry left to start over.)

per Liter:

Agar 20 g + 620 mL water. Autoclave

Meanwhile, make:

Component Volume (mL)
4X SC-AHLT 250
10X 20% glucose 100
50X Tryptophan 20
100X Histidine HCl 10

Add dry:

Component g
YNB 1.7
Adenine hemi sulfate 0.2
Ammonium Sulfate 5

Filter sterilize, warm up, then add to molten agar

Mark with one green line

SC -Leu High Ade 4X

SC -Leu High Ade 4X kdorfman Sun, 01/26/2020 - 16:23

Using dilution factor 4:
(Because 10x won't go into solution)

But the agar is so viscous that bubbles don't reach the surface. Hard to pour.

To make
SC-leu high ade agar plates
with mm plate diameter
pour mL per plate
start with: mL water
add: g agar, leave stir bar in
autoclave for: minutes
Meanwhile, using X SC-AHLT
Asceptically mix together,
warm up, then add to autoclaved agar:		 mL SC-AHLT,
and : mL 10x YNB+AS ,
and: mL 20% glucose ,
and: mL 10X Adenine hemi sulfate,
and: mL 50X Tryptophan,
and: mL 100X Histidine HCl.
for a final volume of mL

Mark with one green line

SC-L high Ade from concentrate

SC-L high Ade from concentrate kdorfman Fri, 01/22/2021 - 20:55
To make SC -L high Ade plates (100 mm)
mix: g agar
into: mL water
autoclave for: minutes
while it's still hot, asceptically add: mL heated 8X SC-L high Ade concentrate
for a final volume of: mL. Mix thoroughly before pouring plates

Mark with one green line.

SC-L high Ade mostly powder

SC-L high Ade mostly powder kdorfman Tue, 02/04/2020 - 13:41

High concentration agar is difficult to work with.

  • Tends to boil over, so needs a lot of water in the autoclave pan
  • Hard to get into solution - frequently needs re-autoclaving
  • So viscous that bubbles don't rise to the surface

So

  • mix the ingredients that can't be autoclaved in as little water as possible
  • Only use high concentration stock solutions for micro-ingredients

Per Liter:

dry ingredient g
SC-AHLT 1.64
YNB 1.7
Ammonium sulfate 5
glucose 20
stock solutions mL
50X Tryptophan 20
100X Histidine HCl 10
10X Adenine hemi sulfate 100

plus water to 150 mL

filter sterilize

Autoclave

  • 20 g agar
  • 850 mL water

Mix and pour

SC -Leu-low-Ade

SC -Leu-low-Ade kdorfman Thu, 01/16/2020 - 22:07

SC-Leu low-Ade plates for Jeff Laney's cell and molecular biology lab

Laney's original

Laney's original kdorfman Sun, 01/26/2020 - 17:02

SC-Leu low-Ade plates

From Jeff Laney's recipe:

(BUT, can't make 10X SC-AHLT)

Per liter of medium, mix the following asceptically, and pre heat:

Component Volume (mL)
10X SC-AHLT 100
10X YNB+AS 100
10X 20% glucose 100
10X Adenine hemi sulfate 2.5
50X Tryptophan 20
100X Histidine HCl 10

Add to 20g agar in 667.5 mL water, autoclaved 30 minutes

Mark with one green and one blue line.

SC -Leu-low-Ade 4X + powder

SC -Leu-low-Ade 4X + powder kdorfman Sun, 01/26/2020 - 17:02

SC -Leu low Ade 4x + powder

Use the 4x SC-AHLT to mix dry ingredients

(4x already made; not enough dry left to start over.)

per Liter:

Agar 20 g + 617.5 mL water. Autoclave

Meanwhile, make:

Component Volume (mL)
4X SC-AHLT 250
10X Adenine hemi sulfate 2.5
10X 20% glucose 100
50X Tryptophan 20
100X Histidine HCl 10

Add dry:

Component g
YNB 1.7
Ammonium Sulfate 5

Filter sterilize, warm up, then add to molten agar

Mark with one green and one blue line

SC -Leu-low-Ade 4X

SC -Leu-low-Ade 4X kdorfman Sun, 01/26/2020 - 17:01

Using 4X SC-AHLT because 10x won't go into solution:

(BUT: Agar is too viscous - bubbles won't rise to the surface before it sets up.)

To make SC-leu low ade agar plates
with mm plate diameter
pour mL per plate
start with: mL water
add: g agar, leave stir bar in
autoclave for: minutes
Meanwhile, using X SC-AHLT
Asceptically mix together,
warm up, then add to autoclaved agar:		 mL SC-AHLT,
and : mL 10X YNB+AS,
and: mL 20% Glucose,
and: mL 10x adenine hemi sulfate,
and: mL 50X Tryptophan
and: mL 100X Histidine .
for a final volume of mL

Mark with one green and one blue line.

SC-AHLT low Ade mostly powder

SC-AHLT low Ade mostly powder kdorfman Tue, 02/04/2020 - 13:26

High concentration agar is difficult to work with.

  • Tends to boil over, so needs a lot of water in the autoclave pan
  • Hard to get into solution - frequently needs re-autoclaving
  • So viscous that bubbles don't rise to the surface

So

  • mix the ingredients that can't be autoclaved in as little water as possible
  • Only use high concentration stock solutions for micro-ingredients

Per Liter:

dry ingredient g
SC-AHLT 1.64
YNB 1.7
Ammonium sulfate 5
glucose 20
stock solutions mL
10X Adenine hemi sulfate 2.5
50X Tryptophan 20
100X Histidine HCl 10

plus water to 150 mL

filter sterilize

Autoclave

  • 20 g agar
  • 850 mL water

Mix and pour

Mark with one green and one blue line.

SC-AHLT

SC-AHLT kdorfman Wed, 01/15/2020 - 21:45

SC minus (adenine, histidine, leucine, tryptophan)

Sunrise Science 1330-030

4X will dissolve. Almost completely clear after overnight stirring. Crystal clear after autoclaving.

From the manufacturer:

Commonly added to YNB, nitrogen and glucose, at suggested g/L, for a complete yeast media. Mix with stirring for 10-15 minutes, and filter sterilize or autoclave at 121°C for 15 minutes. To prepare plates, autoclave agar separately and add to sterile medium, or add agar to liquid medium and adjust to pH 5.8-6.0 before autoclaving.

Suggested g/L: 1.64

Storage Temperature: 2-8 C

Jeff's recipe: 10X SC –AHLT 16.4 g/L

Dissolve 16.4 g of Sunrise Science SC –Adenine, –Histidine, –Leucine and –Tryptophan in 1 L distilled water and filter sterilize.

Store in the dark at 4 ˚C.

Will not go into solution.

SC-HLT Ade-free Concentrate

SC-HLT Ade-free Concentrate kdorfman Wed, 02/02/2022 - 19:18

Concentrate to make SC-L High Ade or Low Ade agar

Make this concentrate, then add adenine hemi-sulfate.

for low-Ade, add 5 mg Ade per L of concentrate

for high-Ade, add 1.6 g Ade per L of concentrate

to make high-Ade from low-Ade concentrate, add 1.595g Ade per L, or 0.1595g per 100 mL

Then filter sterilize. For every L of medium, combine 20g agar autoclaved in 875 mL water with 125 mL warmed up concentrate.

add 125 mL concentrate to autoclaved 875 mL water + 20 g agar

to make enough concentrate for:
Liter(s) SC-L high Ade or SC-L low Ade agar medium
start with
mL water
add:
g YNB
and:
g ammonium sulfate
and:
g SC -HLT
autoclave for:
minutes (to get it into solution)
while it's still hot, add:
g glucose
and:
g tryptophane
and:
mL his 100X concentrate.
Bring to a final volume of:
mL 8X SC-HLT Ade-free. Add the appropriate amount of Ade and filter sterilize

SC-complete-high-Ade

SC-complete-high-Ade kdorfman Wed, 01/15/2020 - 22:12

Liquid Medium

Using 4X SC-AHLT (because 10x won't go into solution):

To make mL SC-complete high ade liquid medium
Meanwhile, using X SC-AHLT
Mix: mL 10X SC–AHLT,
and : mL 10X YNB+AS,
and: mL 20% Glucose,
and: mL 10x adenine hemi sulfate,
and: mL 50X Tryptophan ,
and: mL 100X Histidine .
and: mL 10X Leucine

Bring to final volume with water, then filter sterilize.

Jeff Laney's original (discontinued - because you can't make 10X SC-AHLT) recipe:

Component Volume (mL)
10X SC–AHLT 25
10X YNB+AS 25
10X SC–AHLT 25
20% Glucose 25
10x adenine hemi sulfate 25
50X Tryptophan 5
100X Histidine 2.5
10X Leucine 25
distilled water to final volume of 250

Filter sterilize

Store at room temperature

SC -Leu High Ade

SC -Leu High Ade kdorfman Sun, 01/19/2020 - 13:33

Recipe from Jeff Laney. (But 10X SC-AHLT wouldn't go into solution. See 4X recipe.)

Component Volume (mL)
10X SC-AHLT 100
10X YNB+AS 100
10X 20% glucose 100
10X Adenine hemi sulfate 100
50X Tryptophan 20
100X Histidine HCl 10

Using a different dilution factor:

To make
SC-leu high ade agar plates
with mm plate diameter
pour mL per plate
start with: mL water
add: g agar, leave stir bar in
autoclave for: minutes
Meanwhile, using X SC-AHLT
Asceptically mix together,
warm up, then add to autoclaved agar:		 mL SC-AHLT,
and : mL 10x YNB+AS ,
and: mL 20% glucose ,
and: mL 10X Adenine hemi sulfate,
and: mL 50X Tryptophan,
and: mL 100X Histidine HCl.
for a final volume of mL

Mark with one green line

YEAD

YEAD kdorfman Fri, 08/04/2023 - 22:03

Yeast Extract Adenine Dextrose Medium

Ade mutants grow well on this without turning red. Better for keeping the parental strains white longer.

1 gram Yeast Extract 2 grams anhydrous dextrose (glucose) 2 grams Agar 8 mL adenine stock solution (1 mg/mL)

Adenine Stock Solution

400 mg adenine in 400 ml water (1 mg/ml) Store at room temperature. Use 2 ml stock solution for 100 ml of medium; reduce the water added to the medium by 2 ml.

Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.

To make YEAD agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter-sterilized 1 mg/mL adenine
add asceptically mL filter sterilized 20% glucose
for a final volume of mL

YED

YED kdorfman Mon, 02/08/2016 - 19:22

Yeast Extract Dextrose

For Bio 284

Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.

To make YED agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
for a final volume of mL

Mark with a single red line.

YEKAC

YEKAC kdorfman Mon, 02/08/2016 - 19:29

Sporulation Medium for Bio 284

To make YEKAC agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g potassium acetate, stir till dissolved
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes

OR

  • 20.4 mL 5M KOAc per liter
  • 12.75 mL 8M KOAc per liter Fisher AAJ63372AE

Mark with a single black line.

YEPAD

YEPAD kdorfman Mon, 08/07/2023 - 17:05

YEPAD medium (Yeast Extract + Peptone + Adenine + Dextrose) contains the same ingredients as YEAD but has peptone added. This is a very rich medium used for storing yeast strains.

1 gram Yeast Extract 2 grams Peptone 2 grams anhydrous dextrose (glucose) 2 grams Agar (agar-agar; gum agar) 8 mL adenine stock solution 92 ml water

To make YEPAD agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g yeast extract, stir till dissolved
add: g peptone, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter-sterilized 1 mg/mL adenine
add asceptically mL filter sterilized 20% glucose
for a final volume of mL

YEPAD for freezing

YEPAD for freezing kdorfman Wed, 08/16/2023 - 18:06

A Dohlman Lab Protocol

Yeast strains, transformed or untransformed, can be maintained as colonies on solid media at 4° and restreaking every 2 to 4 weeks. Alternatively, strains may be stored at -80 indefinitely. This is preferable in that it reduces the likelihood of accumulating spontaneous mutations.

To make frozen stocks of yeast strains: -Use sterile technique and sterile solutions throughout this method.-

  1. Grow a starter culture at 30 with shaking (250 rpm) until it reaches saturation.

  2. In a 1.8 ml cryotube, mix 0.5 ml of the saturated culture with 0.5 ml of YPD containing 20% glycerol.

  3. Flash freeze the tube in liquid nitrogen and store at -80.

  4. To use, chip out a few pieces of the frozen stock using a sterile pipette tip or sterile toothpick and streak onto a plate containing the appropriate solid media.

  5. Allow the yeast to grow at 30 until colonies appear (2-6 days).

YNB + AS

YNB + AS kdorfman Wed, 01/15/2020 - 21:44

10X YNB+AS

17 g/L YNB and 50 g/L AS

Dissolve 17 g Difco Yeast Nitrogen Base w/o Amino Acids and w/o Ammonium Sulfate and 50 g Ammonium Sulfate in 1L distilled water.

Autoclave for 15 min.

Store at room temp.

YPD

YPD kdorfman Mon, 02/08/2016 - 15:04

Yeast extract-Peptone-Dextrose medium:

To make YPD agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g peptone broth, stir till dissolved
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
for a final volume of mL YPD agar

Dilutions

Dilutions kdorfman Mon, 03/07/2016 - 21:07

Enter initial and final concentration (in matching units!), and final volume

c1 = stock concentration
c2 =concentration you're trying to make
v2 =volume you're trying to make
v1= Start with this volume, and add water (or other diluent) to final volume

Fixatives

Fixatives kdorfman Tue, 06/16/2009 - 17:53

How to fix cells and tissues

Fixing adherent cells

Fixing adherent cells kdorfman Thu, 05/21/2020 - 17:39

To fix the cells grown on a coverslip

  • Work in a fume hood
  • Gently rinse cells 2X in warm PBS (remove medium, add PBS, repeat)
  • Leave cells in this PBS—until you add the fixative.
  • Remove final PBS rinse, add about 1 mL of fixative to the cells
  • Fix cells in hood for 10 mins.
  • Rinse cells by dunking each coverslip in 3 beakers of PBS-Tween-Azide, 10 X per beaker.
  • Place coverslip back in dish with fresh PBS-Tween-Azide
  • If you are not staining immediately, store cells in PBS-Tween-Azide in fridge.

Remember:

  • Work in the fume hood
  • Wear gloves!
  • Keep track of which side of the coverslip the cells are on
  • Discard used fixative in the appropriate hazardous waste container in the hood.

Formaldehyde fix

Formaldehyde fix kdorfman Thu, 07/05/2012 - 20:55

Make in fume hood 3.7% formaldehyde
0.5% Triton X
in PBS

Formaldehyde stock: 37%
Triton X stock: 10%

To make mL fixative
add: mL 37% formaldehyde
add: mL 10X PBS
and: mL 10% Triton-X

To fix the adherent cells (still in hood):

Methanol fix

Methanol fix kdorfman Tue, 07/24/2012 - 17:05

100% methanol at ice temperature

10 min

No permeabilization step needed.

Wash with PBS afterwards.

The methanol fixation is an easy method; however, it frequently solubilizes and removes membrane bound antigens. By a simple precipitation of the protein, methanol only provides low structural preservation.

PFA 5% for fish

PFA 5% for fish kdorfman Wed, 10/02/2019 - 16:56

5% paraformaldehyde
in PBS

Paraformaldehyde stock: 32%

To make mL fixative
at: % paraformaldehyde
add: mL 32% paraformaldehyde
add: mL 10X PBS

Paraformaldehyde Fix

Paraformaldehyde Fix kdorfman Sun, 10/27/2013 - 19:32

3.7% paraformaldehyde, 0.5% Triton-X
in PBS

Paraformaldehyde stock: 32%
Triton X stock: 10%

EMS RT 15714

To make mL fixative
add: mL 32% paraformaldehyde
add: mL 10% triton-X
add: mL 10X PBS

To fix the adherent cells (still in hood):

Paraformaldehyde from powder

Paraformaldehyde from powder kdorfman Wed, 03/03/2021 - 20:12
to make: mL
at a concentration of: percent
start with: mL 10X PBS
then add: mL water
add and stir: g paraformaldehyde powder

Stir, heat to ~60C

Add NaOH until it clears, then bring to final volume

Paraglut

Paraglut kdorfman Mon, 09/24/2012 - 16:12

3.2% paraformaldehyde
0.1% glutaraldehyde
0.5% Triton X-100

in PBS

To make mL fixative
add: mL 37% paraformaldehyde
add: mL 10X PBS
and: mL 10% Triton-X
add: mL 1glutaraldehyde

Bring to final volume with distilled water.

To fix adherent cells (still in hood):

Gels

Gels kdorfman Thu, 05/24/2012 - 17:17

EtBr gel

EtBr gel kdorfman Tue, 09/20/2022 - 15:02

Ethidium bromide gel

gels:
%:
TAE: mL
agarose: g
EtBr: µL

*Add weighed agarose to measured TAE in a flask (about half the maximum volume of the flask)

*Boil in the microwave CAREFULLY (power level 0.5) until completely dissolved (check by swirling) DO NOT LET IT BOIL OVER

*Allow to cool slightly

*Add EtBr (in the fume hood)

*Aliquot into 50 mL conical tubes

*Put in rack in 65C waterbath

Clean-up

  • Rinse gel rigs within an inch of their lives

  • Put gels into zip lock bag, stick a hazardous waste sticker on it, schedule hazardous waste pickup (sign into CEMS)

  • pour used buffer into gallon jug with EtBr removing "tea bag", stir overnight, then pour down the drain

Sybr Safe gel

Sybr Safe gel kdorfman Tue, 09/20/2022 - 15:01

1 uL /10 mL gel

gels:
%:
TAE: mL
agarose: g
SYBR: µL

*Add weighed agarose to measured TAE in a flask (about half the maximum volume of the flask)

*Boil in the microwave CAREFULLY (power level 0.5) until completely dissolved (check by swirling) DO NOT LET IT BOIL OVER

*Allow to cool slightly

*Add SYBR-safe

*Aliquot into 50 mL conical tubes

*Put in rack in 65C waterbath

Clean-up

Everything can go in the trash or down the drain

Hormones & Inhibitors, etc.

Hormones & Inhibitors, etc. kdorfman Wed, 06/20/2018 - 21:25

By compound

By compound kdorfman Mon, 04/23/2018 - 17:49

ATP

ATP kdorfman Mon, 04/23/2018 - 17:50

10 mM ATP Stock
(from Maniotis)

  • 60 mg ATP in 8 mL H2O
  • pH to 7.0 with 0.1 M NaOH
  • Bring volume to 10 mL with H2O
  • Aliquot and freeze.

Angiotensin

Angiotensin kdorfman Thu, 04/26/2018 - 17:10

Sigma A9525 1mg/mL in water

Vasoconstrictor

Soluble in water 25 mg/mL (only use sterile water!)

MW: 1046.18 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

Subject to degradation in freezer storage - wear gloves!

Make 1 mg/mL stock (= 1.0462 mM)

Working concentration 1 µM

Dissolve 1:1000 in HBS

sigmaa9525.pdf

BMS

BMS kdorfman Fri, 03/25/2022 - 13:50

BMS 833923

Cayman Chemical 16240

___ ___
CAS Registry No. 1059734-66-5
Formal Name: N-[2-methyl-5-[(methylamino)methyl]phenyl]-4-[(4-phenyl-2-quinazolinyl)amino]-benzamide
Synonym XL 139
MF: C30H27N5O
FW: 473.6
Purity: ≥98%S
Stability: ≥2 years at -20°C
Solubility 0.1 mg/ml in a 1:7 solution of ethanol:PBS (dissolve in ethanol first!) Use aqueous solution within 1 day
Supplied as: A crystalline solid
UV/Vis. λmax: 315 nm

Smoothened (Smo) is a cell surface receptor that, with Patched, mediates sonic hedgehog (Shh) signaling to regulate gene expression through the Gli transcription factors.1 BMS 833923 is an orally bioavailable inhibitor of Smo.2,3 It blocks binding of BODIPY cyclopamine (IC50 = 21 nM) and inhibits Gli activation in cell lines that express wild-type Smo or activated mutant forms of Smo (IC50s = 6-35 nM). BMS 833923 robustly inhibits Shh pathway activity and prevents tumor growth in medulloblastoma and pancreatic carcinoma xenograft models.

References

  1. Ruiz-Gómez, A., Molnar, C., Holguín, H., et al. The cell biology of Smo signalling and its relationships with GPCRs. Biochim. Biophys. Acta1768(4), 901-912 (2007).

  2. Sandhiya, S., Melvin, G., Kumar, S.S., et al. The dawn of hedgehog inhibitors: Vismodegib. J. Pharmacol. Pharmacother.4(1), 4-7 (2013).

  3. Lin, T.L. and Matsui, W. Hedgehog pathway as a drug target: Smoothened inhibitors in development. OncoTargets and Therapy5, 47-58 (2012)

BMS Product Information

BP-A

BP-A kdorfman Mon, 03/22/2021 - 18:11

Bisphenol A

Sigma 239658

MW 228.29

100 mM stock in DMSO = 0.023g/mL

BP-S

BP-S kdorfman Mon, 03/22/2021 - 18:12

Sulfonyldiphenol

Sigma 103039

MW = 250.27

100 mM stock in DMSO = 0.025g/mL

Blebbistatin

Blebbistatin kdorfman Mon, 04/23/2018 - 17:49

Myosin inhibitor

(-)-Blebbistatin Sigma B05650-1mg

Info from Cayman:

A stock solution may be made by dissolving the (±)-blebbistatin in an organic solvent purged with an inert gas. (±)-Blebbistatin is soluble in organic solvents such as DMSO and dimethyl formamide (DMF). The solubility of (±)-blebbistatin in these solvents is approximately 10 mg/mL.

(±)-Blebbistatin is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, (±)-blebbistatin should first be dissolved in DMF and then diluted with the aqueous buffer of choice. (±)-Blebbistatin has a solubility of approximately 0.5 mg/ml in a 1:1 solution of DMF:PBS (pH 7.2) using this method. We do not recommend storing the aqueous solution for more than one day.

(±)-Blebbistatin is a selective cell-permeable inhibitor of non-muscle myosin II ATPases.1,2 It rapidly and reversibly inhibits Mg-ATPase activity and in vitro motility of non-muscle myosin IIA and IIB for several species (IC50 = 0.5-5.0 μM), while poorly inhibiting smooth muscle myosin (IC50 = 80 μM).3 Through these effects, blebbistatin blocks apoptosis-related bleb formation, directed cell migration and cytokinesis in vertebrate cells. Blebbistatin is inactivated by UV light,4 which may be particularly important in fluorescent cell imaging applications.

Note green fluorescent crystals seen in a 75µM solution.

Stock was 1 mg/mL in DMSO

44 µL 1mg/mL blebbistatin in DMSO in 1 mL non-CO2 medium

REDO

1 mg in 34 µL DMSO = ~100mM

MW = 292

Use at ~150 µM

blebbistatin-info.pdf

Bradykinin

Bradykinin kdorfman Mon, 04/23/2018 - 18:56
  • 9-amino acid peptide chain: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Bradykinin raises internal calcium levels

*Sigma B3259 - 1 mg septum bottle

  • MW = 1060.2

    1 mg/mL stock
    (= 1.0602 mM)

Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)

Aliquots are 21.2 µL.

2 µM solution for use
(=2.12 µg/mL)

Brefeldin A

Brefeldin A kdorfman Tue, 11/12/2019 - 20:48

Fisher 00-4506 1000x solution

Keep in refrigerator

Description

Brefeldin A is an inhibitor of intracellular protein transport. Incubation of cells in culture with Brefeldin A leads to blockade of protein transport to the Golgi complex (GC) and accumulation of proteins in the endoplasmic reticulum (ER). Addition of Brefeldin A during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines. Brefeldin A is effective for enhanced detection of a majority of mouse and human intracellular cytokines; however, it is advised that the investigators evaluate the use and efficacy of this reagent as well as other protein transport inhibitors such as Monensin in their specific assay system.

Applications Reported

The Brefeldin A Solution is supplied at 1000X working concentration in methanol. Prior to addition to the cell cultures, a 1:1000 dilution in the culture media should be made. The final working concentration of the Brefeldin A (at 1x) is 3.0 ug/ml.

Brefeldin product information

Caffeine

Caffeine kdorfman Tue, 04/13/2021 - 15:25

MW 194.19

Soluble 10 mg/mL in water (with heat and stirring)

Make a 50mM (=0.05M) solution: 9.71mg/mL

Cannabinol

Cannabinol kdorfman Fri, 05/11/2018 - 16:11

Tetrahydrocannabinol solution 1 mg/mL in methanol

Sigma T464

store at 4C

Vial is in a white box in fridge door, room 362A

t4764_data_sheet.pdf

Centrinone

Centrinone kdorfman Thu, 06/01/2023 - 18:38

PLK4 (Polo-like kinase) inhibitor

Plk4 has been identified as a master regulator of centriole replication, and its aberrant expression is closely associated with cancer development

PLK4 functions downstream of ROCK2 to drive centrosome amplification in arrested cells. PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.

100uM stock

  • use 125 nM for HeLa (2.5 uL stock in 2 mL medium)

  • use 300 nM for LLCPk-1 (6 uL stock in 2 mL medium)

Concanavalin A

Concanavalin A kdorfman Mon, 04/23/2018 - 18:21
  • Sigma C5275 5 mg
  • Plant mitogen
  • Lectin (carbohydrate-binding protein) extracted from the jack-bean, Canavalia ensiformis

  • Con A binds specifically to α-Mannose, α-Galactose structures found in sugars, glycoproteins and glycolipids

  • Make stock 5 mg/mL solution (add 1 mL sterile PBS to bottle – gently rotate to mix)

  • Keep sterile - work in sterile hood

  • aliquot 100 µL into microfuge tubes  (cannot take repeated freeze-thaw cycles)

concanavalin_a.pdf

Con A coverslips

Con A coverslips kdorfman Sun, 05/24/2020 - 15:38

Requirements:   * Acid washed coverslips stored in 100% ethanol.

  • ConA 0.5 mg/mL. 

  • Large petri dishes to store the coverslips.    

  Procedure:

  • Flame the acid-washed coverslips and cool for 10 sec.    
    (Make 9-14 coverslips at a time to save time.)

  • Place the coverslips on top of the large petri dish.     

  • Thaw the conA from the freezer and dilute to 0.5 mg/mL 

  • apply around 400uL to the first two coverslips 

  • Wait for 10 sec. 

  • Tilt the coverslips and suck out the excess ConA 

  • Repeat the same for all the coverslips. 

  • Cover the petri dish with another lid of petri dish and allow a small opening to dry.

  • If possible,use a vacuum pump  to dry it faster.   

The process usually takes 15 minutes and drying takes half an hour. 

Storage:

  * In a petri dish, place the coverslips on top of a clean kimwipe with the conA surface facing up.

  • Cover with the lid. 10 min before needed, UV sterilize them.

  • Alternatively, UV sterilize all of them after drying and open the dishes only in aseptic conditions or inside hood.       

Con A dishes

Con A dishes kdorfman Sun, 05/24/2020 - 15:37

Concanavalin-coated coverslip dishes

  • Start with 5 mg/mL ConA stock solution

  • Keep sterile - work in sterile hood

  • aliquot 100 µL into microfuge tubes  (cannot take repeated freeze-thaw cycles)

  • Coat coverslip bottom dishes with either:

    • 0.75 mL 0.1 mg/mL:  0.015 mL (5 mg/mL) + 0.735 mL PBS
    • 0.75 mL 0.5 mg/mL:  0.075 mL (5 mg/mL) + 0.675 mL PBS

  • Incubate the coverslip dishes for 1 hour 37˚C 

  • Remove the solution

  • rinse in PBS 

  • Air dry the dish.

Cyclin Inhibitor

Cyclin Inhibitor kdorfman Wed, 04/25/2018 - 19:55

CKD1 inhibitor IV RO3306

1 mM in DMSO

A cyclin-dependent kinase inhibitor protein inhibits cyclin-dependent kinase. Several function as tumor suppressor proteins. Cell cycle progression is negatively controlled by cyclin-dependent kinases inhibitors (called CDIs, CKIs or CDKIs). CDIs are involved in cell cycle arrest at the G1 phase.

biologics33.2_eusd.pdf

Cycloheximide

Cycloheximide bcrcstaff Thu, 03/21/2019 - 13:28

Protein synthesis inhibitor

Fisher AC357420010

MW = 281.35 g/mol

C15H23NO4

Solubility in water: 2.1 g/100mL (

soluble in chloroform, ether, acetone, methanol, and ethanol

powder

Cyclopamine

Cyclopamine kdorfman Fri, 03/25/2022 - 13:43

ApexBio A8340

10 mM

Store in freezer

C27H41NO2

MW = 411.62

Soluble 6.86 mg/mL in DMSO (warm to 37C and shake to get it into solution)

Reaction Conditions :

  • 20 μM, 48 hours for cell yield inhibition
  • 10 μM, 48 hours for apoptosis induction (measured by PARP expression)

Cyclopamine is a naturally occurring Hedgehog (Hh)?specific small?molecule signaling steroidal alkaloid inhibitor, causes a profound inhibition of tumor growth, has significant anti?invasive, anti?proliferative and anti?estrogenic potency in human breast cancer cells [2] [1]. The EC50 of cyclopamine is 10.57 μM, it was identified by an FXR-bla (farnesoid X receptor- b-lactamase) assay [3].

Hh signaling pathway plays a critical role in embryonic development and tumorigenesis [4]. Hh signaling pathway shows saliency in regulating cellular proliferation and differentiation in a wide array of human tissues. It is related to aberrant cell survival in numerous human malignancies, ranging from BCCs and medulloblastomas to small cell lung, gastrointestinal, breast and prostate tumors [1].

Treated with cyclopamine (10 or 20 μM) only and incubated for time periods ranging from 0 to10 days, MCF-7 cells and MDA?MB?231 cells displayed a significant reduction in proliferation rate compared with the control cells on days 3 and 6 (P

Embryoes exposed to cyclopamine resulted in visible external defects, including cyclopia, proboscis formation, microphthalmia, thoracic lordosis, amelia and decreased body size. Examination of gastrointestinal organs revealed severe deficits, including less length of the gut tube and mesenchymal cell numbers in foregut-derived organs. Ectopic structures in duodenum, stomach, and dorsal pancreas were also found [5].

Cytochalasin D

Cytochalasin D kdorfman Mon, 04/23/2018 - 18:21

MW = 507.62
Sigma C2618: 200 µL 5 mg/mL (~10 mM) in DMSO
Sigma C8273: 5 mg. Dissolve in 1 mL DMSO

  • alkaloid mycotoxin produced by Helminthosporium and other molds.
  • disrupts actin microfilaments
  • binds to F-actin polymer
  • prevents polymerization of actin monomers
  • Aliquot 10 µL 10 mM
  • Add 90 uL DMSO (makes 1 mM)
  • Dilute this 1:1000 into medium (makes 1uM)

  • Dilute the 1 uM to desired working concentration

  • Store in the dark, in the freezer

  • Adding 490 µL of non-CO2 medium makes 500 µL of 200 µM

  • Final dilution should contain no more than 0.1% DMSO

  • 250 nM in the dish should give an effect

  • 15 - 30 min

c2618dat.pdf

DAPT

DAPT kdorfman Fri, 03/25/2022 - 18:16

Biological Activity for DAPT (Info from Tocris 2634)

Purchased from Fisher: AAJ65864MA

Mfr: Thermo Scientific Chemicals J65864MA

M. Wt: 432.46 Formula: C23H26F2N2O4 Store at +4°C Light sensitive

Solubility: 43.24 mg/mL (100 mM) in DMSO

DAPT is a γ-secretase inhibitor. DAPT reduces Aβ40 and Aβ42 levels in human primary neuronal cultures (IC50 values are 115 and 200 nM for total Aβ and Aβ42 respectively) and in brain extract, cerebrospinal fluid and plasma in vivo. DAPT has no effect on APPα and APPβ levels. DAPT blocks Notch signaling in hybrid human-mouse fetal thymus organ culture (FTOC) and causes ESCs to commit to neuronal differentiation. DAPT can be used in a small molecule cocktail to derive cortical neurons from hPSCs and to maintain hepatocytes in culture. DAPT also promotes the formation of cone photoreceptors in retinal organoids.

Tocris DAPT information sheet

Estradiol

Estradiol kdorfman Thu, 03/24/2022 - 17:40

β17-Estradiol

Cayman Chemical 10006315

(Purchased 2022 for NAP lab Bio 388)

500 mg

Store powder at -20

Make 0.05M stock solution: 0.068 g in 5 mL DMSO. vortex

__ __
CAS​ Registry​No 50-28-2
Formal​ Name estra-1,3,5(10)-triene-3,17β-diol
Synonyms β-Estradiol, Estradiol, 17β-Oestradiol, E2
MF C18H24O2
FW 272.4
Purity ≥98%
Stability ≥2 years at -20°C
Supplied ​as A crystalline solid
UV/Vis λmax: 281 nm
solubility 2.5 mg/mL in ethanol
20 mg/mL in DMSO
0.2 mg/mL in a 1:4 solution of DMSO:PBS (pH 7).
Dissolve in DMSO first!
Use within a day

Estrogens direct the development of the female genotype in embryogenesis and at puberty. 17β-Estradiol is the major estrogen secreted by the premenopausal ovary. It is synthesized from testosterone primarily in the ovarian granulosa cells and placenta, but small amounts can be produced in the adrenal gland.1,2 Plasma 17β-estradiol levels increase gradually between days 1-7 of the menstrual cycle followed by a sharp increase to a peak value of about 300 pg/ml on day 12, just prior to ovulation

Fibronectin

Fibronectin kdorfman Thu, 04/26/2018 - 17:15

Sigma F1141

Attachment factor

GSA 10

GSA 10 kdorfman Thu, 01/28/2021 - 18:39

Smo (Smoothened) receptor* agonist

Tocris 4918 10 mg

MW = 459.94

Make a 5 mM stock solution in 4.34 mL DMSO (heat to 50C to dissolve)

*Non-classical G-protein-coupled receptors that belong to the Frizzled family. Smoothened receptors lack the ability to directly interact with their endogenous ligand, Hedgehog (Hh). In the resting state, Smo receptors are bound to patched (Ptc).

Does not recognize the classic cyclopamine (Cat. No. 1623) binding site. Does not promote Smo translocation to the primary cilium; is strongly potentiated by forskolin and cholera toxin. Displays anti-adipogenic effects in vitro, mediated by a non-canonical Hedgehog signaling pathway. Promotes differentiation of multipotent mesenchymal progenitor cells into osteoblasts.

H-89

H-89 kdorfman Mon, 03/22/2021 - 18:08

H 89 hydrochloride Tocris 2910

  • Protein kinase A inhibitor
  • Also inhibits several other kinases (IC50 values are 80, 120, 135, 270, 2600 and 2800 nM for S6K1, MSK1, PKA, ROCKII, PKBα and MAPKAP-K1b).
  • Exhibits antinociceptive activity.
  • Enhances survival and clonogenicity of dissociated human ESCs through ROCK inhibition.

Store in freezer

1 mg

MW 532.79

1 mg/mL in DMSO

data sheet

HU (Hydroxyurea)

HU (Hydroxyurea) kdorfman Wed, 01/06/2021 - 18:29
ingredient working conc MW stock
HU 0.2 M 76 Koshand lab 2M in H2O

Hydroxyurea: (Sigma H8267-1g)

  • To make 50 mL YPD with 0.2M HU

  • vi = (0.2M * 50 mL)/2M = 5 mL

  • 100µL 2M stock per mL medium

  • Buy 1 g, make 6.6 mL 2M stock

  • MW = 76, so 1 g = 1/76 mol = 0.0132 mol
  • vol = (1000 mL/2 mol) * 0.0132 mol = 6.6 mL

See yeast recipes for QBoC https://wahoo.cns.umass.edu/node/554/

Haloperidol

Haloperidol kdorfman Thu, 03/24/2022 - 20:06

TCI H0912

C21H23CIFNO2

MW 375.87

Solubility:1

  • 0.1 M HCl: 3 mg/mL
  • DMSO: soluble
  • H2O: insoluble
  • ethanol: soluble (only in theory - in practice, not so much)

Make 10 mL 10mM stock solution: 0.376g in 10 mL DMSO. Vortex to mix

Haloperidol has been used:

  • in ethanol to serves as an inhibitor of Erg2p
  • to address the mechanism of haloperidol in ferroptosis using hepatocellular carcinoma cells: Hep G2 and Huh-7 cell lines
  • in receptor internalization assay
  • as an antipsychotic drug in Dulbecco′s Modified Eagle medium

Biochem/physiol Actions:

Haloperidol is a butyrophenone antipsychotic. It is also classified as a neuroleptic (powerful tranquilizer). Haloperidol acts as a D2, D3, and D4 dopamine receptor antagonist and thus causes Parkinson′s disorder. It also has a negative effect on the central nervous system.

  1. from Sigma ↩︎

Hydrocorisone

Hydrocorisone kdorfman Mon, 03/22/2021 - 18:06

Fisher A16292 (Alfa Aesar) 1 g

1 mg/mL in 100% ethanol.

IGF-1R I

IGF-1R I kdorfman Fri, 03/25/2022 - 14:37

Insulin-like growth factor-1 receptor inhibitor

Selleck Chemicals BMS-536924

Molecular Formula: C25H26ClN5O3

Formula Weight: 479.96

Solublity: 96 mg/mL in DMSO

IWP-2

IWP-2 kdorfman Fri, 03/25/2022 - 16:47

Selleck Chemical S7085 (Fisher NC0736836)

IWP-2 is an inhibitor of Wnt processing and secretion with IC50 of 27 nM in a cell-free assay, selective blockage of Porcn-mediated Wnt palmitoylation, does not affect Wnt/β-catenin in general and displays no effect against Wnt-stimulated cellular responses. IWP-2 specifically inhibits CK1δ.

Selective inhibitor of Porcn-mediated Wnt secretion.

IWP-2 is useful in both regenerative medicine and anticancer efforts. IWP-2 inactivates Porcn, a membrane-bound O-acyltransferase (MBOAT), and selectively inhibits palmitoylation of Wnt. IWP-2 blocks Wnt-dependent phosphorylation of Lrp6 receptor and Dvl2, and β-catenin accumulation.

Soluble in DMF at 12.5 mg/mL (26.79 mM) Insoluble in water and DMSO

Concentration 1 mg/ 5 mg/ 10 mg/
1 mM 2.1432 mL 10.7158 mL 21.4316 mL
5 mM 0.4286 mL 2.1432 mL 4.2863 mL
10 mM 0.2143 mL 1.716 mL 2.1432 mL
50 mM - - -
Tocris IWP-2 information sheet

Importazole

Importazole kdorfman Thu, 05/10/2018 - 14:25

401105-10mg Calbiochem

Importazole is an inhibitor of importin-β transport receptors.

During interphase, the transport receptor importin-β carries cargoes into the nucleus, where RanGTP releases them. Importazole somehow disrupts the importin/RAN interaction. Importazole is selective among other transporters. Compounds are imported into, but not out of, cells.

A cell-permeable diaminoquinazoline compound that selectively blocks importin-β-mediated nuclear import of NLS bearing cargos in a reversible manner.

Soluble in DMSO (25 mg/ml; clear, colorless solution)

5 mg/mL ailquots in freezer

Ionomycin

Ionomycin kdorfman Mon, 04/23/2018 - 18:47

Raises intracellular calcium levels

Fisher or Invitrogen (Life Technologies) I24222

1 mM in DMSO or EtOH. 1 mg makes 1.34 mL stock.

3 µM Ionomycin in HBS
Dilute stock 1:333 in HBS: 3 µL/1 mL

24 µL in 8 mL, aliquot 800 µL

They only need 750µL, because they do one experiment in HBS, then two in Ca-free HBS.

3 µM Ionomycin in Ca-free HBS
Dilute stock 1:333 in Ca-free HBS: 3 µL/1 mL
45 µL in 15 mL

Takes a long time to thaw. Vortex.

Aliquot 1.55 mL so they can do two 750 µL experiments

Jasplakinolide

Jasplakinolide kdorfman Thu, 04/26/2018 - 17:44

(out of stock 2018)

  • Jasplakinolide – Santa Cruz biochemical, product sc202191 F
    • inducer of actin polymerization & stabilization; inhibits actin filament disassembly
    • F-actin probe
    • MW = ~710
    • powder is stable frozen over a year
    • stock is stable 3-4 mo at -20C
    • Make 1 mM stock with 50 µg in 70 µL DMSO
    • Add 5 mL non-CO2 medium to make ~14 µM
    • 50 nM to 5 μM working range
    • minutes to hours incubation time

LDL

LDL kdorfman Fri, 05/11/2018 - 16:29

Low-density lipoprotein from human plasma, Dil complex (Dil LDL)

1 mg/mL

200 uL

DO NOT FREEZE

Life Technologies L3482

Laminin

Laminin kdorfman Thu, 04/26/2018 - 18:16

Laminin from mouse BD354232

1 mL 1 mM

Cell adhesion

sigma_laminin_l2020.pdf

Latrunculin

Latrunculin kdorfman Thu, 04/26/2018 - 17:46

Alexis Biochemicals 350-036-C100 Catalog # t110

Inhibits actin polymerization and disrupts microfilament organization, as well as microfilament-mediated processes

Reported to be 10 to 100-fold more potent than the cytochalasins. May act more slowly, though. Lots of variation between cell lines.

Inactivated by FBS. (Rinse medium off before treatment.)

MW = 395.5

Soluble in DMSO and ethanol.

Store in freezer.

Active concentration range: 90 nm ( =~0.1µM) to 2.5 µM

(2.5 µM = 25 x 100 nm)

Stock is 1mm

µL 1mM stock µL serum-free buffer or medium µM final concentration
1 * 99 10
1 399 2.5
1 999 1

*Use this to make further dilutions

latrunculin_data_sheet.pdf

ML-7

ML-7 kdorfman Wed, 04/25/2018 - 19:42

ML-7 Sigma, 12764.

selective myosin light chain kinase inhibitor

  • soluble 10mg/mL in 50% EtOH
  • MW = 452.74
  • 5 mg in bottle
  • add 1100 µL to make 10 mM solution
  • used at ~50 µM (1 µL/1mL) (Pat says 75 µM)
  • 75 µL 10 mM + 5 mL non-CO2 medium makes 150 µM
  • make 15 µL aliquots of 10 mM. Add 985 µL medium to make 150 µM
i2764sigma.pdf

Melanocyte Stimulating Hormone

Melanocyte Stimulating Hormone kdorfman Wed, 04/25/2018 - 20:48

Sigma M4135 1 mg

Molecular Weight 1664.88

From Sigma: Amino Acid Sequence:

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2

Pituitary hormone which causes darkening skin pigmentation from amphibians to humans. In mammals, it can also have behavioral effects on learning, attention, and memory.

Hormone that stimulates melanogenesis; facilitates learning and memory; affects inflammatory and immune responses and peripheral nerve regeneration.

α-Melanocyte-stimulating hormone (α-MSH) acts as an anti-inflammatory agent via down regulating the production and activity of the pro-inflammatory cytokines interleukin-1 (IL-1), tumor necrosis factor (TNF)-α and IL-6 expressed in various cells of the immune system. It also controls the nitric oxide production associated with inflammation. α−MSH inhibits nuclear factor-κB (NF-κB)-dependent gene transcription and NF-κB pathway induced by TNF and other inflammatory agents. This activity of α-MSH is mediated through the production of cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) enzyme. α–MSH functions as a potent therapeutics for various conditions resulted through NF-κB activation including, inflammatory diseases, human immunodeficiency virus (HIV) replication in AIDS (acquired immunodeficiency syndrome), and septic shock.[2] α-MSH has an essential role to play in melanin production in animals. α-MSH regulates development of several skin diseases, including cutaneous inflammation and hyper-proliferative skin diseases.[3] Linkage

Derived from ACTH 1-13 Other Notes

Lyophilized from 0.1% TFA in H2O Packaging

1, 5 mg in glass bottle Application

α-Melanocyte stimulating hormone (α–MSH) has been used for following studies: • it has been Intracerebroventricularly (icv) injected to mice for behavioral studies.[1] • to determine the effect of α−MSH on growth of stationary Nb 2 node lymphoma cell cultures.[4] • to determine the effect of α−MSH on leptin secretion in the primary cultures of differentiated adipocytes.[5] • α−MSH promoted melanin production in the B16-F1 cells from murine melanoma cell line.[6] General description

α-Melanocyte-stimulating hormone (α-MSH) is a tridecapeptide, mostly produced by the cells in the brain, pituitary and circulation.[2] Pro-inflammatory cytokines or UV light induced epidermal cells such as keratinocytes and melanocytes synthesize and discharge α–MSH. Poopiomelanocortin (POMC) acts as a precursor for α-Melanocyte-stimulating hormone (α-MSH) production.[3]

m4135sigma.pdf

Monastrol

Monastrol kdorfman Thu, 05/10/2018 - 15:17

Inhibits kinesin 5

200 mM in DMSO

Final concentration 200 uM

5.5 uL aliquots

Monensin

Monensin kdorfman Wed, 04/25/2018 - 21:25

Monensin sodium salt

Sigma M5273

soluble in methanol 50 mg/mL

insoluble in water

soluble in DMSO, ethanol

ionophore which disrupts the structure of the Golgi apparatus and inhibits vesicular transport in eukaryotic cells

inhibits transition from G1 to S

antiprotozoal, antibacterial, or antifungal agent

MW 692.9

1 µM = half-maximal inhibitory effect

monensin.pdf

Nerve Growth Factor

Nerve Growth Factor kdorfman Fri, 05/11/2018 - 16:16

Nerve Growth Factor 2.5S

Harlan.com Cat no. 5025

From Sigma: 2.5S subunit of nerve growth factor-7S (NGF-7S) is essentially the β-subunit when isolated from male mouse submaxillary glands under initially dissociative conditions by a modification of the method of Bocchini and Angeletti.

Stored at 4C

Nocodazole

Nocodazole kdorfman Mon, 04/23/2018 - 17:51

Prevents polymerization of microtubules. Prevents cells from entering metaphase.

  • Acros 358240100, 10 mg

  • Soluble in DMSO

  • Dilute stock in medium to treat cells.

  • Make a 10 mg/mL (=33mM) stock: Add 1 mL DMSO to 10 mg in bottle.

  • Aliquot 3 µL 33 mM per tube.

  • Working concentration range is usually 100nM - 30 µM.

  • add 97 µL DMSO to tube to make 100 µL of 1 mM

  • 33 µL 1mM + 10 mL medium = 3.3 µM

Oxidopamine

Oxidopamine kdorfman Fri, 03/25/2022 - 15:41

Oxidopamine (hydrobromide)

Selleck Chemicals S5324-25mg (Fisher 17159569)

MW = 250.09

Formula: C8H11NO3.HBr

Oxidopamine (6-hydroxydopamine, 6-OHDA, 2,4,5-trihydroxyphenethylamine) is a neurotoxic synthetic organic compound that acts as an antagonist of the neurotransmitter dopamine with potential antineoplastic activity. Solutions should be freshly prepared and protected from exposure to light.

solvent mg/mL mM
DMSO 50 mg/mL (199.93 mM)
Ethanol 50 mg/mL (199.93 mM)
Water 50 mg/mL (199.93 mM)
Concentration 1 mg/ 5 mg/ 10 mg/
1 mM 3.9986 mL 19.9928 mL 39.9856 mL
5 mM 0.7997 mL 3.9986 mL 7.9971 mL
10 mM 0.3999 mL 1.9993 mL 3.9986 mL
50 mM 0.0800 mL 0.3999 mL 0.7997 mL

Plant hormones

Plant hormones bcrcstaff Thu, 03/21/2019 - 14:49
Compound function Form Room storage stock Cat #
ABA Plant hormone involved in growth and stress response powder 362A fridge 50 mM in EtOH Fisher AC133480010
ABA powder soluble 20 mg/mL in DMSO, ethanol 362A freezer PlantMedia 30631017-1
soluble in methanol
Ascorbic acid Enzyme cofactor Powder 362A RT Fisher S26184
Caffeine Growth inhibitor Powder 362A RT Fisher ICN15011483
Chitosan medium viscosity Component of fungal cell walls and arthropod exoskeletons Powder 362A RT Bio-World 40300161-1
Cycloheximide Protein synthesis inhibitor Powder 362A RT Fisher AC357420010
Gibberellic acid breaks seed dormancy 13 mg/mL (=37.5mM) solution 362A 4C PlantMedia 30631025-1
IAA auxin growth regulator
stimulates root growth		 solid 362A 4C 100 mM in EtOH PlantMedia 30631010-
Imidacloprid (pestanal) Systemic insecticide neat 362A dessicator Sigma 37894
kinetin Plant cytokinin Powder 362A RT Sigma K0753
Lactic acid Plant growth stimulator 69% soln aq 362A dessicator Fisher AC250300100
Methyl jasmonate Pathogen response Liq 362A RT PlantMedia30631015-3
Nicotine Plant defense chemical liq 362A dessicator Fisher AC181420050
Salicylic acid Response to abiotic stress Powder 362A RT 100 mM in EtOH Fisher S25515
Selenium varies Powder 362A RT Strem 93-3416

Poly-L-Lysine

Poly-L-Lysine kdorfman Thu, 04/26/2018 - 17:04

Non-specific attachment factor for cells; use it to coat coverslips.

Sigma P1524

  1. Add 50 ml of sterile tissue culture grade water to 5 mg of poly-lysine.
  2. Aseptically coat culture surface with 1 ml per 25 cm2 of solution. Rock gently to ensure even coating of the culture surface.
  3. After 5 minutes, remove solution by aspiration and thoroughly rinse surface with sterile tissue culture grade water.
  4. Allow to dry at least two hours before introducing cells and medium. If glassware or slides must be sterilized after coating with poly-lysine, γ-irradiation is recommended instead of autoclaving.

Can also buy poly-lysine coated slides: Fisher 6776215

sigmap1524.pdf

Purmorphamine

Purmorphamine kdorfman Thu, 01/28/2021 - 18:25

Tocris 4551 10 mg

MW = 525.12

Make a 10 mM stock in DMSO in 1.9 mL DMSO

Retinol

Retinol kdorfman Wed, 04/27/2022 - 17:09

Fisher J62079

Store in freezer; hygroscopic

C20H30O

MW = 286.459

General description (from Fisher)

  • All-trans-retinol, commonly known as Vitamin A, is a fat-soluble essential nutrient
  • All-trans-retinol is involved in hormonal signaling via the retinoid receptors
  • It is an important regulator of cell division and apoptosis
  • Deficiency can lead to vision, bone, immune, and skin disorders

Practically insoluble aq. soln. or glycerol; soluble in absolute alcohol, methanol, chloroform,ether,fats and oils.

Can make a 50 mg/mL solution in 95% ethanol, but it's very hard to weigh

From Sigma information sheet:

SOLUBILITY / SOLUTION STABILITY:

  • RE is practically insoluble in water or glycerol. It is soluble in absolute ethanol, methanol, chloroform, ether, fats and oils.
  • RE has been dissolved at 50 mg/ml in chloroform; a clear yellow to orange solution results.
  • Stock solutions of RE (1 mg/ml) were prepared in ethanol, diluted in DMSO under low light conditions and stored at -50°C under nitrogen in brown glass vials.
  • RE solutions (50 μM) were sterile filtered before use.
  • RE both as a solid and in solution is readily oxidized in air and inactivated by UV light.
  • To reduce photodestruction of RE, manipulations of RE solutions can be performed under yellow or red light.
  • Solutions may be stabilized by dissolving in oil, by the addition of anti-oxidant compounds including a-tocopherol or hydroquinone or by conversion to the palmitate and acetate esters.
  • It is recommended to prepare solutions fresh for optimal quality. However, if absolutely necessary, store solutions in the dark under an inert atmosphere at least at -20°C preferably at -70°C
  • Solvents preferred for storage are peroxide-free ethyl ether, acid-free acetone or ethyl acetate.
  • For short term storage, ethanol is suitable as a solvent for spectroscopic analysis.

USAGE / APPLICATION

  • The isolation of retinol from human plasma has been described.
  • RE is an effective antioxidant displaying lipoperoxy radical scavenging activity.
  • The interactions between RE and Vitamin E (a-tocopherol) in suppressing lipid peroxidation were observed in bovine retinal membrane preparations.
  • RE may influence the production of transition vesicles by stimulating the activity of a protein disulfide isomerase-like activity involved in vesicle formation.
  • RE may be involved in immune system mechanisms; an RE deficiency will depress the immune response producing a negative effect on both humoral and cellular immunity.
  • (10 μM) and other retinoid compounds effectively induced sanguinarine and chelerythrine (benzophenanthridine alkaloids) accumulation in suspension-cell cultures of Sanguinaria canadensis in a way similar to fungal elicitation.
  • RE (10 μM) stimulated DNA synthesis and possibly repair mechanisms in Sertoli cells of rat.

GENERAL NOTES

The USP unit of vitamin A (same as the International Unit6) is equal to 0.3 μg of the pure all-trans isomer of retinol which is equivalent to 0.344 μg of all-trans retinyl acetate. RE and its metabolites, including retinoic acid, are part of the retinoid class of compounds, involved in vision, normal embryo morphogenesis and in the regulation of proliferation and differentiation of a number of cell types. Current information and hypotheses on the absorption, transport, storage and metabolism of this fat soluble Vitamin A (retinol) have been reviewed. Studies on RE metabolism including its mobilization and transport in plasma and in tissues via serum and cytosolic retinol-binding proteins have been described.

STLC

STLC kdorfman Mon, 04/23/2018 - 17:51
  • STLC Sigma 164739 (Fisher 50-703-1833), MW = 363.47

(+)-S-Trityl-L-cysteine is a cell-permeable selective inhibitor of mitotic kinesin Eg5 and ATPase activities.

KIF11 (also known as kinesin-5 and Eg5) is a homotetramer which cross-links anti-parallel microtubules in the mitotic spindle to maintain spindle bipolarity. The motor domain or motor head is at the N-terminus and performs ATP hydrolysis and binds to microtubules. (https://en.wikipedia.org/wiki/Kinesin_family_member_11)

  • Make 100 mM stock:

    • 1 g in bottle.
    • Mix with 27.5 mL DMSO
    • heat to 65C (~10 min), vortex
    • if necessary, filter sterilize to remove insoluble particles.
    • save as 1 mL aliquots in freezer. (dilute 1:10,000 to use) (dilute 1:100 to make 1 mM)

  • Make 1 mM stock from the 100 mM stock

    • 10 µL 1mM stock
    • 990 µL DMSO
    • vortex
    • it will crystallize in the refrigerator. Warm (60C) and vortex to redissolve it. DO NOT LEAVE IN HOT BLOCK - IT WILL TURN BLACK!

  • Make 10 µL aliquots of 1 mM stock. Freeze

    • label says to add 990 µL medium to make 10 µM working solution
164739sigma.pdf

Sodium alginate

Sodium alginate kdorfman Thu, 04/28/2022 - 14:52

ALGINIC ACID SODIUM SALT 5G

Fisher 177770050 $22.41

(Can get 100g from Sigma for $55, but with long lead time)

(C6H7O7)A(C6H7O7)BNa

MW=120,000-190,000 g/moL

The following is from Sigma:
General description

Alginic acid sodium is a gelling and nontoxic anionic polysaccharide. The carboxylic acid groups on the alginic acid chain, renders it insoluble in water.However, converting alginic acid to its sodium form, enables it to solubilize in water easily.

Application

Alginic acid sodium is used:

  • in combination with chitosan, to fabricate a biodegradable porous scaffold for bone tissue engineering.
  • to study the characteristics of a modified amphiphilic alginate derivative
  • to the study the impact of alginate on the rate of lipid digestion by employing an in vitro digestion model
  • in the preparation of alginate hydrogels
  • as encapsulating agents of microparticles of β-galactosidae

THC

THC kdorfman Fri, 04/22/2022 - 17:40

Restek 34067

Delta-9 THC Standard

1 mg/mL in methanol (=~3.18 mM)

C21H30O2

MW = 314.469

Taxol

Taxol kdorfman Mon, 04/23/2018 - 17:51

Taxol (20 µM final)

Fisher 109710 Paclitaxel.
Sigma T7402 1mg
MW = 853.91

  • Stabilizes the microtubule polymer
  • Protects microtulues from disassembly.
  • Prevents metaphase spindle configuration

  • Blocks progression of mitosis and prolonged activation of the mitotic checkpoint triggers apoptosis or reversion to the G-phase of the cell cycle without cell division.

  • soluble in DMSO, not water.

  • Add 0.117 mL DMSO to make a 0.01M (=10 mM) stock solution from the 1 mg powder

  • Aliquot 10 µL 10 mM (if it precipitates, add 10 uL DMSO and vortex, then add 480 uL medium or buffer)

    • add 490 µL to make 200µM
    • dilute into medium from there (100 uL Taxol/mL medium)
taxolsigma.pdf

Thyroid hormones

Thyroid hormones kdorfman Fri, 09/06/2019 - 18:38

for Fish

5 mL per well for treatment

PTU (inhibitor) Sigma p3755
MW 170.2
Solubility:

  • 50 mg/mL in 1M NaOH
  • 16 mg/mL in alcohol
  • 1 mg/mL in RT water
  • 10 mg/mL in boiling water

In order to get the following doses, best to make it in hot fish water; otherwise, the NaOH or EtOH will be too concentrated in the final dilution.

  • dose 1: 0.5 mM

  • dose 2: 1.0mM

MW of PTU:
Concentration: M
to make: mL
add: g PTU

L-T4 (thyroxin) Sigma T2376
MW: 776.87
solubility:

  • 50 mg/ml in 4M NH4OH in MeOH
  • 0.5 mg/mL in 1:5 DMSO:PBS

Solution is good for ~ 1 day.

Make a 1 mM stock in 1M NaOH. Takes a long time. Vortex.

Priya's recipe: 1 mg/mL in 1M NaOH (~25.7 uM stock)

Working concentrations

  • dose 1: 100 nM (=0.1 uM)

  • dose 2: 300 nM (=0.3 uM)

MW of T4:
Concentration: M
to make: mL
add: g T4

PTU-Sigma-p3755.pdf
Thyroxine_T2376-Sigma.pdf
L-Thyroxine (Cayman)

Trichostatin A

Trichostatin A kdorfman Wed, 04/25/2018 - 19:52

Cayman 89730

10 mM

Histone deacetylase Inhibitor

Trichostatin A (TSA), an antifungal antibiotic produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of histone deacetylases (HDACs), which are overexpressed in various cancers and closely correlate with oncogenic factors.

Trichostatin A is active at nanomolar concentrations in mammalian cells. By suppressing the activity of HDACs, it leads to increased histone acetylation, thereby causing highly acetylated histones to accumulate in the cell [2]. This in turn induces enhanced expression of specific genes that elicit extensive cellular morphologic and metabolic changes such as growth arrest, differentiation and apoptosis. At submicromolar concentrations Trichostatin A has been shown to induce apoptosis in diverse cancer cells while exhibiting very low toxicity to normal cells.

TSA is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, TSA should be directly dissolved in 0.1 M HCl (1.3 mg/ml) and then neutralized with PBS (pH 7.2) to achieve the desired concentration or pH. Approximately 0.7 ml of PBS (pH 7.2) is required to neutralize 1 ml of the acidic solution. We do not recommend storing the aqueous solution for more than one day.

trichostatin89730.pdf

Valinomycin

Valinomycin kdorfman Wed, 04/25/2018 - 21:03

Potassium Ionophore, MW: 1111.32

Sigma V0627

Insoluble in water

stock is 10 mg/mL in DMSO

  • cyclododecadepsi-peptide ionophore antibiotic.
  • potassium ionophore that transports K+ across biological and artificial lipid membranes.
  • can induce K conductivity in cell membranes
  • uncouples oxidative phosphorylation,
  • induces apoptosis in murine thymocytes, and in pre-B cell.
  • inhibits NGF-induced neuronal differentiation
  • antagonizes ET-induced vasoconstriction.
  • Useful in studies of K transport in mitochondria.
valinomycin.pdf

Valproate

Valproate kdorfman Fri, 03/25/2022 - 15:34

Sodium Valproate (Fisher 11474361)

Store powder at RT

Make 100 mM stock: 0.166g in 10 mL H2O

___ ___
SKU 02152064-CF
Alternate Names 2-Propyl pentanoic acid; Valproic acid sodium salt; Sodium 2-propylpentanoate
Application Notes Sodium Valproate is reported to cause inositol depletion, activate the ERK pathway, inhibit GSK-3α and GSK-3β. Valproic Acid has been reported to be a potent inhibitor of HDAC (histone deacetylase) in vitro (IC50 = 400 μM for HDAC1), thereby relieving HDAC-dependent transcriptional repression and causes the hyperacetylation of histones in cultured cells. In animal studies, Valproic Acid has been observed to reduce tumor growth and metastasis formation. Additionally, Valproic Acid is reported to activate Wnt-dependent gene expression and to mimic trichostatin A in the inhibition of histone deacetylase. This compound is also an inhibitor of the CYP2C9 enzyme.
Base Catalog Number 152064
Biochemical Physiological Actions Anti-convulsant that also has efficacy as a mood stabilizer in bipolar disorder.
Boiling Point 219.5 deg C
CAS # 1069-66-5
Density 0.904 g/cu cm at 25 deg C
EC Number 213-961-8
Format Crystalline powder
Hazard Statements H302
Molecular Formula C8H15NaO2
Molecular Weight 166.196 g/mol
Personal Protective Equipment Dust mask , Eyeshields, Gloves
RTECS Number YV7876000
Safety Symbol GHS07
Solubility In water, 2.0X10+3 mg/L at 20 deg C
Usage Statement Unless specified otherwise, MP Biomedical's products are for research or further manufacturing use only, not for direct human use. For more information, please contact our customer service department.
Vapor Pressure 8.47X10-2 mm Hg at 25 deg C (est)

Vasopressin

Vasopressin kdorfman Mon, 04/23/2018 - 17:50

(=Anti-diuretic hormone)

causes calcium oscillations

1 mM stock in water
(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)

1 mg in bottle, MW = 1084
Add 0.92 mL to make 1mM stock

1 µL aliquots in freezer

1 µM dilution to make student solutions

1 µL 1 mM in 999 µL water

(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)

Vitamin D

Vitamin D kdorfman Fri, 04/22/2022 - 17:53

Thermo Scientific B22524.03

D3-calciferol

C27H44O

MW = 384.65

Solubility (from Cayman Chemical)

solvent mg/mL = mM
ethanol 30 ~78 mM
DMSO 3 ~ 7.8 mM
DMF 25 ~65 mM

Stock solution: 29 mg/mL (~75 mM) in ethanol

General Description (from Thermo Scientific)

  • Vitamin D3 is a steroid hormone generated in the skin when the 7-dehydrocholesterol interacts with ultraviolet irradiation. It can also be found in several types of food for daily intake.
  • Vitamin D3 binds to vitamin D receptors, thus modulating gene expression

Applications

  • In in vivo studies, vitamin D3 has been implicated in the maintenance of blood calcium and phosphorus levels, bone metabolism, metabolic functions, and transcription regulation
  • It modulates the proliferation and differentiation of both normal and cancer cells
  • It presents in vitro antiproliferative and antimetastatic activities on breast, colon, and prostate cancer cells

Primers

Primers kdorfman Fri, 05/31/2013 - 17:35

Fisher Custom Oligos

  • Primers come lyophilized.

  • Tube label says how many nmol in the tube (usually ~100-500)

  • Multiply nmol x 10 = μL of sterile water to add to make 100 μM stock. Fisher primer calculator

  • This is the only stock!

  • Put it away safely on instructor shelf in freezer.

  • Make a working stock for students (give them all of it):

  • 12.5 μM (12.5 μL of the 100 μM stock + 87.5 μL water).

(If using the repeater pipet for the water, 85 µL water + 12.14 µL 100 µM stock)

Sea Water

Sea Water kdorfman Thu, 02/16/2017 - 19:51

Instant Ocean

  • 34 g/L
  • pulverize in mortar and pestle
  • add very slowly to the water, stirring
  • autoclaving helps the salts go into solution a little

Artificial Seawater, according to Wikipedia

salt molarity
NaCl 0.409
Na2SO4 0.003
KCl 0.009
NaHCO3 0.0023
KBr 0.00082
H3BO3 0.00042
NaFl 0.00007
MgCl2 0.5327
CaCl2 0.01033
SrCl2 0.00009

Stains

Stains kdorfman Thu, 08/13/2015 - 20:47

Acridine Orange

Acridine Orange kdorfman Fri, 07/27/2018 - 18:06

Sigma A8097

10 mg/mL solution in water

MW = 308.81

Ex: 486 - 492
Em: 516 - 532

From the Sigma web page:

Application

Acridine Orange hydrochloride solution has been used to study autophagic cell death. It has also been used for the staining of chromosomes.

Biochem/physiol Actions

Acridine Orange is a metachromatic dye which can stain DNA, RNA and acid glycosaminoglycans. At low concentration it intercalates into DNA and precipitates RNA. However, at high concentration it denatures and precipitates both RNA and DNA. Acridine Orange is also used to analyze autophagy. It goes into acidic organelles in a pH-dependent manner. At neutral pH, acridine orange gives a green fluorescence and in acidic conditions, it accumulates in the acidic organelle giving a bright red fluorescence.

From use in MBoMS: Use at ~20µM

  • 1.2 µL added to 2 mL in dish

  • incubate 15 min at 37

  • change medium

  • incubate 15 min at 37

  • Replace medium with PBS

  • observe with

    • B excite - G emission: dsDNA
    • G excite - R emission: RNA, ssDNA

Got good results initially, but within minutes, cells started to ball up and pull off the substrate. Will try new PBS first, then a concentration gradient of acridine orange.

Old PBS produced fast shrinking and balling up. Newer PBS was less bad. Now try with non-CO2 medium. Does it interfere with fluorescence?

No cell shrinkage in non-CO2 medium. At short exposure times, there is a background glow, but picking the right exposure takes care of that.

This is time sensitive. The red emission (which should be RNA & ssDNA) gradually fades, or overlies the green emmision (which should be ds DNA)

See attached images, taken in order 1, 2, 3. #3 is at about 15 minutes. 1 & 2 are 100x, 3 is 200x.

Click-iT® Plus EdU Imaging Kits

Click-iT® Plus EdU Imaging Kits kdorfman Mon, 08/12/2019 - 18:55

Materials required but not in kit:

Stock Solutions

Component conc dilute with
A (EdU) 10 mM 2 mL DMSO (C) or buffer
B (Alexa Fluor® picolyl azide)
C (DMSO)
D (reaction buffer) 10X
E Cu protectant
F (buffer additive)
G (Hoechst® 33342)

Per tube, need 200uL

To make mL Click-iT cocktail
add: uL D (reaction buffer)
add: uL Copper protectant (E)
add: uL Alexa Fluor (B)
and: uL F (buffer additive)

Click-iT® Plus EdU Imaging Kits manual.pdf

Buffer Additive

Buffer Additive kdorfman Thu, 03/26/2020 - 14:18

To make a 10X stock solution of the Click-iT® EdU buffer additive (Component F): Add 2 mL deionized water to the vial, then mix until fully dissolved. After use, store any remaining stock solution at ≤–20˚C.

When stored as directed, this stock solution is stable for up to 1 year. If the solution develops a brown color, it has degraded and should be discarded.

EdU

EdU kdorfman Wed, 09/11/2019 - 17:00

33mM (10X) Stock solution

100 mg of EdU powder (purchased from Carbosynth) https://www.carbosynth.com/carbosynth/website.nsf/(w-productdisplay)/D1…

is dissolved in

1.2 mL DMSO, then brought to 12 mL with H2O

Original recipe says: 10.8mL 0.5x E2 medium and

1.2ml DMSO

Rolf says the final concentration of DMSO should be more like 0.1% (after dilution of the 10X EdU to 1X in fish water)

Says to mix with water and warm gently (to thaw, use 60C for 15 minutes)

So try 33 mM EdU in 1% DMSO.

To make 20 mL 10X EdU in 1% DMSO, mix

  • 166 mg EdU
  • 0.2 mL DMSO (heat gently by putting it in a beaker of heated water)
  • bring to final volume with distilled water

Reaction Buffer

Reaction Buffer kdorfman Thu, 03/26/2020 - 14:13

Prepare a working solution of 1X Click-iT® EdU reaction buffer (Component D):

Transfer the solution (4 mL) in the Component D bottle to 36 mL of deionized water.

To make smaller amounts of 1X Click-iT® EdU reaction buffer,

  • dilute volumes from the Component D bottle 1:10 with deionized water.

  • After use, store any remaining 1X solution at 2–8˚C.

  • When stored as directed, this 1X solution is stable for 6 months.

DAPI Fluoromount

DAPI Fluoromount kdorfman Fri, 03/31/2023 - 21:35

DAPI Fluoromount Fisher OB010020

Store at room temp, in the dark

Aliquot 1 mL into dark tube.

Keep in "mounting" drawer in 360.

Make droppers by melting the end of a glass Pasteur pipet to make a ball.

DAPI in glycerol for fish

DAPI in glycerol for fish kdorfman Thu, 02/24/2022 - 19:36

1 mg/mL DAPI stock solution from Rolf

1 uL per 10 mL 25% glycerol

DNA gel stains

DNA gel stains kdorfman Mon, 08/27/2018 - 20:57

3-color LD + SYBR Safe

3-color LD + SYBR Safe kdorfman Mon, 10/15/2018 - 17:38

The orange runs fast; suitable for small DNA fragments

To make 10 mL from home made LD:

  • 6 mL orange G (Fisher AAJ60562AC)
  • 1 mL home made LD with bromophenol blue and xylene cyanacol
  • 6 uL SYBR safe
  • 1 mL glycerol
  • 0.1 mL 1 M Tris pH 7.6
  • water to 10 mL

EtBr

EtBr kdorfman Mon, 08/27/2018 - 21:01

Ethidium bromide

1 uL / 100 mL gel (or DNA sample, if adding it to the loading dye) (=1/100,000)

10 mg/mL
10 mL
17-ETBC1001 Krackeler $37.12

Home-made

Home-made kdorfman Mon, 08/27/2018 - 21:13

6X Home-made loading dye:

  • 30% glycerol
  • 0.3% Bromphenol blue
  • 0.3% xylene cyanol

plus SYBR-Safe at 0.5 uL/1mL

1 uL loading dye stains 5 uL of DNA sample.

SYBR safe works at 1/10,000

need 5/10,000 uL for 5 uL of sample

so need 5/10,000 uL SYBR safe for each uL of loading dye.

= 5/10 uL for each mL

NOTE Fisher Tritrack loading dye (FERR1161) composition:

  • 0.03% bromophenol blue
  • 0.03% xylene cyanol
  • 0.15% orange g
  • 60% glycerol
  • 10 mM Tris, pH 7.6
  • 60 mM EDTA

One tenth as much dye, twice as much glycerol.

SYBR Safe

SYBR Safe kdorfman Mon, 08/27/2018 - 21:04

SYBR safe DNA gel stain

Fisher S33102

400 uL $72.62

Use 1 uL/10 mL gel or DNA sample (=1/10,000)

Safe-Green

Safe-Green kdorfman Mon, 08/27/2018 - 21:13

Safe-Green

(DNA stain + loading dye)

ABM G108-G
1 mL $65.00 ($30 + shipping)

Use at 1:5

Smart-glow

Smart-glow kdorfman Mon, 08/27/2018 - 21:07

Smart Glow loading dye

Krackeler 26510-E4500-LD
1mL $77.22

DNA stain + loading dye.

Add directly to DNA sample

Use at 1:5

DiOC6(3)

DiOC6(3) kdorfman Fri, 05/11/2018 - 16:44

Invitrogen D273

DiOC6(3) is a cell-permeant, green-fluorescent, lipophilic dye that is selective for the **mitochondria:: of live cells, when used at low concentrations. At higher concentrations, the dye may be used to stain other internal membranes, such as the endoplasmic reticulum.

ER Tracker

ER Tracker kdorfman Thu, 04/05/2018 - 15:34

ER Tracker Red

(BODIPY™ TR Glibenclamide), for live-cell imaging Invitrogen/Life/ ThermoFisher: E34250

Ex = 587 nm Em = 615 nm

MW = 915.23

100 ug lyophilized.

To make stock solution:

  • Add 110 uL DMSO to make a 1mM solution
  • Make 1 uL aliquots

To use

Working concentration: ~1 uM.

  • remove growth medium

  • rinse with HBSS

  • Add 1 mL HBSS to 1 uL aliquot to make 1uM solution. (Use buffered saline with Ca++ and Mg++)

  • treat cells with warm 1 uM solution 15 - 30 min

  • rinse staining solution out with warm HBSS

  • put warm non CO2 medium in dish

ertracker.pdf

Fluo-4 AM

Fluo-4 AM kdorfman Thu, 04/26/2018 - 18:04

Fluo-4 stock
Invitrogen F14217 500 µL

Calcium indicator (fluoresces when bound to Calcium ions)

Ex 494 nm; Em 516 nm

1mM in DMSO
Protect from light
Store in dissicator.
20 µL aliquots. Each makes 10 mL of 2 µM solution

Fluo-4 staining solution
2 µM Fluo-4 + 0.02% pluronic in HBS

Incubate 15 - 60 min at 20 - 37C. Wash before viewing.

20 % w/v Pluoronic

plus HBS to final volume

fluo-4.pdf

Lysotracker

Lysotracker kdorfman Thu, 10/24/2019 - 20:17

Lysotracker (Invitrogen L-7528 - 20 x 50 µL)

50 - 75 nM
stock = 1 mM in DMSO
aliquot 10 µL in 2 mL tubes, so they can make 2 mL medium
30 min - 2 hours incubation warm.
Lysotracker protocol

Mitotracker

Mitotracker kdorfman Wed, 02/21/2018 - 18:59
Mitotracker protocol

Mitotracker GREEN

Mitotracker GREEN kdorfman Thu, 10/24/2019 - 20:34

Thermo fisher M7514

Excitation 490 nm : Emission 516 nm

MW: 674

50 ug in vial.

Add 74 uL DMSO to make 1 mM stock.

working concentration =~ 25 - 200 nM

1 uL 1mM stock into 10 mL medium makes 100 nM treatment solution

Mitotracker RED

Mitotracker RED kdorfman Thu, 10/24/2019 - 20:34

Invitrogen M7512

https://www.thermofisher.com/order/catalog/product/M7513?SID=srch-srp-M…

Ex = 579 nm; Em = 599

50 ug per tube.

MW = 531

Add 100 uL DMSO to 50 ug in tube. Makes ~ 1 mM stock solution.

Use at 25 - 100 nM. (1 uL per 10 mL for 100 nM)

Dilute in medium. Treat with pre-warmed stain solution for 15 - 45 minutes. Replace staining solution with fresh (warm) medium.

To fix: use 3.7% formaldehyde (in medium); 37C for 15 minutes (??)

NBD Ceramide

NBD Ceramide kdorfman Wed, 04/25/2018 - 14:32

Invitrogen N22651

fluorescent marker for Golgi in live cells

Ex: 466nm Em: 536nm

Follow instructions attached: Add 150 µL sterile H2O to the 5 mg in the bottle. (Makes 0.5mM (=500 µM) in BSA)

Aliquot 10 µL, and freeze

Working concentration is 5 µM

Add 990 µL HBSS (Hank's)(0.34 mg/mL BSA) to 10 µL in tube

Makes enough for 8 groups.

Aliquot ~120 µL per group, enough to cover the small circular coverslip in the viewing dish.

To use:

  • Rinse with HBSS (see notes)
  • Treat with ceramide solution
  • incubate in cold 30 min
  • rinse 3x in cold HBSS
  • replace HBSS with non-CO2 medium, incubate ~20 minutes

NOTES:

ceramide.pdf

Nuc Blue

Nuc Blue kdorfman Thu, 07/14/2022 - 18:02

Hoechst type DNA stain for live cells

Nuc Blue protocol from Thermo Fisher

  • Culture cells in an appropriate medium and vessel for fluorescence microscopy.
  • Add two drops (20 uL = 1 drop) of NucBlue Live ReadyProbes Reagent per milliliter of medium.1
  • Incubate for 20 minutes, protected from light.
  • Image the cells.

  1. In some cases, more or fewer drops may be needed to achieve optimal staining intensity. Image quality may be improved by replacing the culture medium with Live Cell Imaging Solution (Cat. No. A14291DJ). [Kate says start with less, and work up. Rinse cells after incubation.] ↩︎

Nucleus-RFP

Nucleus-RFP kdorfman Fri, 10/27/2023 - 18:57

CellLightTM Nucleus-RFP

Invitrogen (Thermo-Fisher) C10603

1 mL - received 10/26/23 - purchased for Bioimaging

Store in refrigerator

manual

Simplified protocol:

Starting concentration = 108 particles per mL

Working concentration = 10 - 50 particles per cell

Use low-passage number cells;

estimate number of cells at time of treatment - should be no more than 70% confluent

(number of cells x ~30 particles/cell)/108 particles/mL = mL CellLight to use

Mix thoroughly but gently with cell medium

Image cells after about 16 hours

To stain cells
at particles per cell (between 10 & 50)
add µL Nucleus-RFP

From the mfgr:

CellLight Nucleus-RFP, BacMam 2.0, provides an easy way to label nuclei with red fluorescent protein (RFP) in live cells. Simply add the reagent to your cells, incubate overnight, and the cells are ready to image in the morning.

This ready-to-use construct is transfected into cells using BacMam 2.0 technology, where it expresses RFP fused to the SV40 nuclear localization sequence. You can observe nucleus-RFP behavior in live cells without the cellular toxicity associated with intercalators and label with multiple tracking or tracing dyes to image dynamic cellular processes.

Cells expressing CellLight constructs can also be fixed with formaldehyde for multiplexed imaging using immunocytochemical techniques.

CellLight Technology is:

  • Fast and convenient: simply add CellLight reagent to your cells, incubate overnight, and image—or store frozen, assay-ready cells for later use
  • Highly efficient: up to 90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
  • Flexible: co-transduce more than one BacMam reagent for multiplex experiments or co-localization studies; tightly control expression levels by simply varying the dose
  • Less toxic: CellLight reagents are non-replicating in mammalian cells and are suitable for biosafety level (BSL) 1 handling

BacMam Technology

CellLight Nucleus-RFP, BacMam 2.0, is a fusion construct of SV40 nuclear localization sequence and TagRFP, providing accurate and specific targeting to cellular nucleus-RFP. This fusion construct is packaged in the insect virus baculovirus, which does not replicate in human cells and is designated as safe to use with biosafety level (BSL) 1 in most laboratories. BacMam technology ensures that most mammalian cell types are transduced/transfected with high efficiency and minimal toxicity. This transient transfection can be detected after overnight incubation for up to five days—enough time to carry out most dynamic cellular analyses. Like any transfection/transduction technique, the BacMam method does not transfect/transduce all of the cells with equal efficiency, making it poorly suited to cellular population studies or automated imaging/counting. CellLight reagents are ideal for experiments where cellular or subcellular co-locatization is required, or for cellular function studies that need special resolution.

Visualize staining your cell without wasting your reagents, antibodies, or time with our new Stain-iT Cell Staining Simulator.

PS-Speck

PS-Speck kdorfman Thu, 02/01/2018 - 13:43

Fluorescent Beads

(Should also try MultiSpeck beads M 7901)

Protocol:

  • use polylysine slides so beads stick
  • 5 uL beads total (can mix colors on one slide)
  • 12 uL water; mix well
  • dry on a slide warmer (or hot plate at about 50C)
  • draw a circle on the bottom of the slide around the dried material
  • put 1 drop mounting medium over dried spot (gloppy! can't pipet!)
  • coverslip
  • nail polish

Molecular Probes P7220

Code Color Ex (nm) Em (nm) filter cube label
A blue 360 440 UV
B green 505 515 B
C orange 540 560 LP (?) G?
D deep red 633 660 probably not
PS-Speck Beads Manual.pdf
MultiSpeck Beads manual

Phalloidin

Phalloidin kdorfman Thu, 08/13/2015 - 21:03

General protocol:

Stock Solution: Add 1.5 mL MeOH to vial (~6.6µM)

Aliquots

  • 5 µL stock per 0.5 mL tube
  • Label: Add 200 µL PBS (or PBS 1% BSA), use 50µL per coverslip

Fix cells with formaldehyde

Procedure:

  • 50 uL phalloidin on parafilm in humid chamber
  • coverslip cell side down onto drop
  • 15 - 20 minutes at room temp
  • rinse repeatedly in PBS-Tween-azide
  • blot the corner on a kimwipe
  • Mount on a drop of mounting medium on a slide
phalloidin_labels.docx
phallotoxins.pdf

FITC phalloidin

FITC phalloidin kdorfman Fri, 06/01/2018 - 18:40

Thermo Fisher F432

From the manufacturer:

Fluorescein phalloidin is a high-affinity F-actin probe conjugated to the green fluorescent dye, fluorescein (FITC).

  • Selectively stains F-actin
  • Excitation/Emission: 496/516 nm
  • Superior to antibody staining
  • Optimal for fixed and permeabilized samples

10 and 20 uL aliquots

In histology freezer

Red phalloidins

Red phalloidins kdorfman Fri, 06/01/2018 - 18:42

Alexa Fluor 568 Phalloidin

Alexa Fluor 568 Phalloidin kdorfman Fri, 06/01/2018 - 18:41

Alexa Fluor 568 Phalloidin

Fisher or Invitrogen A 12380

Binds to F-Actin

Ex/Em 578/600

Stock Solution: Add 1.5 mL MeOH to vial (~6.6µM)

Aliquots

  • 8 µL stock per 0.5 mL tube
  • Label: Add 400 µL PBS (or PBS 1% BSA), use 50µL per coverslip

Fix cells with formaldehyde

Procedure:

  • 50 uL phalloidin on parafilm in humid chamber
  • coverslip cell side down onto drop
  • 15 - 20 minutes at room temp
  • rinse repeatedly in PBS-Tween-azide
  • blot the corner on a kimwipe
  • Mount on a drop of mounting medium on a slide

In histology and bioimaging freezers

Rhodamine Phalloidin

Rhodamine Phalloidin kdorfman Fri, 06/01/2018 - 18:35

Thermo Fisher R415

From the manufacturer:

Rhodamine phalloidin is a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine (TRITC).

  • Selectively stains F-actin
  • Excitiation/Emission: 540/565 nm
  • Superior to antibody staining
  • Optimal for fixed and permeabilized samples
  • Very widely cited fluorescent phalloidin conjugate

In 262A freezer

Texas Red-X Phalloidin

Texas Red-X Phalloidin kdorfman Fri, 06/01/2018 - 18:30

Thermo Fisher T7471

From the manufacturer:

Texas Red®-X phalloidin is a high-affinity F-actin probe conjugated to our bright, photostable, red fluorescent Texas Red®-X dye.

  • Selectively stains F-actin
  • Outstanding fluorescence performance
  • Excitation/Emission: 591/608 nm
  • Superior to antibody staining
  • Optimal for fixed and permeabilized samples

In histology freezer

Phloroglucinol

Phloroglucinol kdorfman Wed, 03/17/2021 - 16:10

Stains lignin

Phloroglucinol-HCl (Wiesner) Staining

Dissolve 0.3 g of phloroglucinol in 10 mL absolute ethanol to prepare a 3% phloroglucinol solution.

Mix one volume of concentrated HCl (37 N) to two volumes of 3% phloroglucinol in ethanol; this solution is phloroglucinol-HCl (Ph-HCl) or Wiesner stain. May 13, 2014

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4186213/

3% Phloroglucinol solution

to make: mL
at: %
mix: g phloroglucinol in 100% ethanol

Ph-HCL stain

to make: mL phloroglucinol stain
mix: mL 3% Phloroglucinol solution
with: mL HCl

Syto RNASelect

Syto RNASelect kdorfman Wed, 04/25/2018 - 19:47

SYTO RNASelect Green Fluorescent Cell Stain (Invitrogen S32703)

$210 from Invitrogen

Fluoresces green when bound to RNA.

excite: 490 nm, emit: 530 nm

Can be used in live or fixed cells. Fix in methanol, NOT formaldehyde!

Don't use in conjunction with red-orange dyes.

Stock:

100µL 5 mM in DMSO. Store at -20C, dessicated, dark.

To thaw: warm to RT, spin down.

Should be stable for >= 1 year.

Make labeling solution

Make 5µM intermediate stock:

2 µL 5mM stock + 1998 µL medium or PBS

Make 20 100 µL aliquots in 1.5 mL tubes

Label: RNASelect - 100 µL - 5 µM intermediate stock in PBS; Add 900 µL to make labeling solution

Make 500 nM labeling solution in medium or PBS

100 µL 5 µM intermediate + 900 µL medium

Protocol

Live cells

  • Cells on coverslip
  • Warm 500 nM labeling solution to 37C
  • Incubate at 37C 20 min
  • Rinse twice in PBS or medium
  • Add warm medium, let cells rest 5 min
  • Fix in chilled methanol 10 min at -20C
  • Several washes in PBS

Fixed cells

  • Remove coverslip from medium
  • Fix in chilled methanol 10 min at -20C
  • Remove methanol, let slip sit in PBS 5 min
  • Apply labeling solution 20 min RT
  • Wash 5 min in PBS
  • Mount coverslip
syto_rnaselect.pdf

TMRE

TMRE kdorfman Wed, 04/25/2018 - 21:17

Tetramethrylrhodamine ethyl ester perchlorate

mitochondria-specific Red fluophore

Biochemika (Sigma) 87917

Soluble in DMSO, alcohols.

Stock is 50 mM in DMSO

λex 540 nm; λem 595 nm in DMSO

cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria.

Potential-sensitive probe for measuring membrane potential changes in mitochondria

In freezers in 266A and 362A

Tetramethylrhodamine α-Bungarotoxin

Tetramethylrhodamine α-Bungarotoxin kdorfman Fri, 06/01/2018 - 19:06

Thermo Fisher T1175

Binds to acetylcholine receptor at neuromuscular junction

From the manufacturer:

Tetramethylrhodamine a-bungarotoxin can be used to visualize this receptor. Labeled a -bungarotoxin conjugates can be used to facilitate identification of nicotinic AChRs and to localize neuromuscular junctions.

Excitation⁄Emission (nm): 554⁄577

In freezer 266A

bungarotoxin.pdf

Toluidine blue

Toluidine blue kdorfman Wed, 03/17/2021 - 16:12

Toluidine blue is a basic thiazine metachromatic dye with high affinity for acidic tissue components.

It stains nucleic acids blue and polysaccharides purple and also increases the sharpness of histology slide images. It is especially useful today for staining chromosomes in plant or animal tissues

Toluidine blue for Madelaine's plant anatomy class

  • 1 % Toluidine blue
  • 0.5% borax

Toluidine Blue Stock Solution:

1g in 100 mL 70% ethanol

NaCl acid solution

1% NaCl aqueous, pH between 2 - 2.5 (w/ glacial acetic acid)

Working toluidine solution

1 part toluidine stock solution : 9 parts NaCl-Acid

Make this solution fresh and discard after use. pH higher than 2.5 will make staining less contrast.

To make: mL working toluidine blue solution,
mix: mL 1% NaCl + glacial acetic acid, pH ~2.25
with: mL 1% toluidine blue in 70% ethanol

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahU…

Transferrin

Transferrin kdorfman Thu, 05/10/2018 - 14:49

Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM).

Final concentration = 1 µM.

Aliquot 2.5 µL.

Add 150 µL Fe-HBS-BSA to make 153 µL 1 µM

Store in freezer

Transferrin is a monomeric serum glycoprotein (~80,000 daltons) that binds up to two Fe3+ atoms for delivery to vertebrate cells through receptor-mediated endocytosis.

Once iron-carrying transferrin proteins are inside endosomes, the acidic environment favors dissociation of iron from the transferrin–receptor complex. Following the release of iron, the apotransferrin is recycled to the plasma membrane, where it is released from its receptor to scavenge more iron.

Fluorescent transferrin conjugates can therefore be used with fluorescent LDL to distinguish the lysosomally directed and recycling endosomal pathways.

transferrin_manual.pdf

Stock solutions

Stock solutions kdorfman Fri, 10/28/2011 - 15:35

ABA

ABA kdorfman Tue, 11/17/2015 - 19:28

(+)-cis,trans-abscisic acid

Plant Media 30631017-1 250 mg $68

soluble in EtOH, MeOH, DMSO 20 - 50 mg/mL

MW = 264.3

Store as powder in freezer.

Make 1 mL of 100mM solution:

0.1mol/1000mL x 164.3g/mol = 0.0264 g

Working concentration ~0.15 mM

In 30 mL MS agar, =

vi = 30mL x 0.15 mM/100 mM = 0.045 mL

ATP

ATP kdorfman Mon, 11/04/2013 - 17:02

10 mM ATP Stock
(from Maniotis)

60 mg ATP in 8 mL H2O
pH to 7.0 with 0.1 M NaOH
Bring volume to 10 mL with H2O

Aliquot and freeze.

Use at ~10 µM

Adenine

Adenine kdorfman Wed, 10/18/2017 - 22:41

Fisher AC147440250 25 gr.

1 mg/mL solution for yeast MV-Ade medium

heat (65C) and stir for one & a half hours

filter sterilize

Adenine hemi-sulfate

Adenine hemi-sulfate kdorfman Wed, 01/15/2020 - 21:39

10X Adenine hemi-sulfate (Sunrise ScienceProducts 1905-010)

2 g/L

Dissolve 2 g of Adenine per 1 L distilled water and filter sterilize. Store in the dark at 4 ˚C.

Antibiotics

Antibiotics kdorfman Wed, 12/28/2011 - 21:01

G418

Table of antibiotic stability in poured agar

Amphotericin

Amphotericin kdorfman Tue, 01/09/2018 - 18:29

Anti-Fungal

Stock solution: 10 mg/mL in DMSO (insoluble in ethanol)

Store at 4C short term, -20 long term (in 15 mL tube, wrapped in foil with tape label)

Keep in the dark.

Final concentration in medium = 10 ug/mL

Add 1 mL per L

Freezes in the refrigerator.

Protect from light.

Ampicillin

Ampicillin kdorfman Wed, 12/28/2011 - 21:02

50 mg/mL stock

(Final concentration in medium = 50 µg/mL)

Lasts up to 4 weeks in poured agar at 4C

0.5 g in 10 mL water

filter sterilize

1 mL aliquots (enough for 1 L medium)

0.4 mg/mL for QSB

Make 35 mL: 0.28 mL 50 mg/mL stock

35 mL sterile water

(filter sterilize if any doubt about sterility)

48 0.7 mL aliquots

Can also use carbenicillin

to make: mL amp solution
at this concentration: mg/mL
add: g dry ampicillin

Carbenicillin

Carbenicillin kdorfman Tue, 08/02/2016 - 15:05

(More stable than ampicillin)

Fisher 50841234b 25 mL Teknova C2130

100 mg/mL stock

effective concentration 50 - 100 µg/mL in medium

Freeze aliquots.

Cipro

Cipro kdorfman Wed, 12/28/2011 - 21:02

Ciprofloxacin 10 mg/mL stock

(final concentration in medium = 10 µg/mL)

100 mg in 10 mL dilute acid (add HCl drop by drop, mixing in between till it dissolves)

filter sterilize

1 mL aliquots

freeze

0.2 mg/mL for QSB

0.7 mL 10 mg/mL stock

35 mL water

filter sterilize

48 0.7 mL aliquots

To make mL 0.2 mg/mL cipro
you'll need: g cipro

freeze

Doxycycline

Doxycycline kdorfman Mon, 01/18/2016 - 13:27

Tetracycline category

solubility 50 mg/mL in water

disk has 30 ug

Erythromycin

Erythromycin kdorfman Fri, 01/20/2012 - 18:10

solubility

50 mg/mL in ethanol

lasts up to 4 weeks in poured agar at 4C

10 mg/mL stock

(10 µg/mL in medium)

0.25 g in 25 mL EtOH

1 mL aliquots

freeze

0.6 mg/mL for QSB

2.1 mL 10 mg/mL stock

33 mL EtOH1

48 0.7 mL aliquots

freeze

  1. Try 20 µL in 330 µL water to see if it dissolves. If so, then make aqueous solution, filter sterilize, and freeze. ↩︎

Kanamycin

Kanamycin kdorfman Wed, 12/28/2011 - 21:01

50 mg/mL stock (50 µg/mL in medium)

Concentration in poured agar drops to ~80% in 1 week, but stays stable for 3 more weeks at 4C

1.25 g in 25 mL H2O

filter sterilize

mL aliquots

freeze

1.2 mg/mL for QSB

0.84 mL 50 mg/mL stock

34.16 mL water

filter sterilize (if any doubt about sterility)

48 0.7 mL aliquots

To make mL 1.2 mg/mL kanamycin
you'll need: g kanamycin

freeze

Kan calcs

Kan calcs kdorfman Tue, 10/02/2012 - 16:23
if Kanamycin stock is: mg/mL
to make: mL medium
with a final Kan concentration of: mg/mL
Add: mL kanamycin stock

Streptomycin

Streptomycin kdorfman Tue, 10/17/2023 - 19:43

In refrigerator in 362A

Fisher Streptomycin sulfate 50g

BP910-50 (discontinued)

MW 1457.58

~50 mg/L for agar

Tetracycline

Tetracycline kdorfman Wed, 01/18/2012 - 20:28

Standard is 10 mg/mL stock

Stable for 2 weeks in poured agar at 4C; 75% after 4 weeks

15 mg/mL stock

0.275 g tetracycline in 25 mL EtOH

1 mL aliquots

freeze

1.2 mg/mL for QSB

2.8 mL 15 mg/mL stock in 32.2 mL EtOH1

48 0.7 mL aliquots

freeze

To make mL 1.2 mg/mL tetracylcine
you'll need: g tetracycline

  1. Try 28 µL in 322 µL water to see if it dissolves. If so, then make aqueous solution, filter sterilize, and freeze. ↩︎

Triclosan

Triclosan kdorfman Wed, 12/28/2011 - 21:02

Irgasan

Sigma 72779-5g-f

MW 289.54

Boric acid

Boric acid kdorfman Tue, 12/20/2011 - 15:09

Boric acid (H3BO3) 0.01 M (=10 mM)

1000x for M&S micronutrients

MW = 61.83

0.031 g/50 mL

MW of Boric acid:
Concentration: M
to make: mL
add: g boric acid

Calcium chloride

Calcium chloride kdorfman Tue, 12/20/2011 - 15:01

CaCl stocks in two concentrations:

MW (dihydrate) = 147

1 Molar

2.205 g/15 mL

7.35 g/50 mL

**Autoclave (or otherwise sterilize) for *C. elegans* medium**

0.5 Molar (=500 mM)

1.1 g/15 mL

3.675 g/50 mL

**Autoclave (or otherwise sterilize) for *C. elegans* medium**

MW:
Concentration: M
to make: mL
add: g CaCl2

Calcium nitrate

Calcium nitrate kdorfman Mon, 08/03/2015 - 18:47

Ca(NO3)2.4H2O

FW = 236.1

0.4M stock:

94.4g/L = 0.189g/50mL

MW of calcium nitrate:
Concentration: M
to make: mL
add: g calcium nitrate

Chelated Iron

Chelated Iron kdorfman Fri, 01/12/2024 - 21:28

FeSO4.7H2O 2.78 g/L
Disodium EDTA 3.73 g/L

Mix each component in 450 mL water

Heat EDTA to boiling

Add EDTA to the FeSO4 solution.

Boil for an hour, cool completely

Bring to final volume

Cholesterol

Cholesterol kdorfman Mon, 01/09/2012 - 17:47

5 mg/mL for C. elegans medium

Fisher AAA1147018 Alfa Aesar 50 g ~$35

in 95% ethanol

Stir for 3 hours

or vortex several minutes

Do not autoclave!

Cobalt Chloride

Cobalt Chloride kdorfman Tue, 12/20/2011 - 15:11

Cobalt Chloride (CoCl2) 105 µM (=~ 0.1 mM)

10,000X for M&S micronutrients

MW (hexahydrate) = 237.93

0.00125 g/50 mL

MW:
Concentration: M
to make: mL
add: g dry stuff

DNA standards

DNA standards kdorfman Wed, 12/22/2021 - 21:38

NEB MARKERS

NEB DNA standards (for general reference)

NEB 1Kb extend

NEB PCR Marker

NEB 100 bp quick load ladder

NEB 1 Kb ladder (we have quick load and regular)

FISHER MARKERS. (See attached pdfs below for pictures of ladders)

Thermofisher DNA ladders (for general reference)

Gene ruler ladders (for general reference)

Fisher Scientific molecular weight markers (for general reference)

GeneRuler 50 bp SM0373

GeneRuler 100 bp SM0243

Gene Ruler Mix SM0331

MassRuler Mix SM0403

MassRuler High MW SM0393

50bp GeneRuler
100bp GeneRuler
GeneRuler Mix
MassRuler Mix
MassRuler High Range

DTT

DTT kdorfman Fri, 05/31/2013 - 17:21

Dithiothreitol

Acros organics426380500 (store refrigerated in 362A) $98 at Fisher

154.25 MW

1M = 1.5425g in 10 mL H20

MW:
Concentration: M
to make: mL
add: g DTT

EDTA

EDTA kdorfman Tue, 12/20/2011 - 14:45

EDTA (disodium) 0.5 M pH 8.0

MW = 372.4

18.62 g/100 mL

9.31 g/50 mL

initial pH = ~6. Use NaOH pellets to bring to pH = 8. ~2 g NaOH/100 mL

MW:
Concentration: M
to make: mL
add: g EDTA

EGTA

EGTA kdorfman Fri, 08/09/2013 - 20:27

EGTA 0.5M

100 ml solution

19g EGTA (MW 380g/mol) ddH2O to 90ml adjust pH 7.5/8.0 with solid NaOH (>4g) adjust volume to 100ml

MW:
Concentration: M
to make: mL
start with: mL
add: g EDTA, adjust pH to 7.5 or 8 with NaOH pellets, then bring to final volume

Note: EGTA will not go into solution without NaOH. Once the pH has been raised sufficiently it dissolves quickly. For pH 7.5 the exact amount required is slightly above 4g. Add 3.5-4g immediately, then proceed carefully not to overshoot the desired pH.

from http://www.researchgate.net/post/How_can_I_dissolve_EGTA

Ferric citrate

Ferric citrate kdorfman Tue, 12/20/2011 - 15:14

Ferric citrate (C6H5FeO7) 89.4 mM

MW=244.95

1.095 g/50 mL

0.328 g/15 mL

Keep refrigerated

The 89.4 value is because Tobias Baskin's lab makes a 89.4 µM Fe-citrate medium, and it's easy to mix up a batch from the 1000x stock solution.

Maximum solubility is 1 g/100 mL hot H2O

MW of ferric citrate:
Concentration: M
to make: mL
add: g ferric citrate

Could in the future make a 10 mM batch with 0.1225 g in 50 mL, and for this lab: https://wahoo.nsm.umass.edu/content/reagents-61 , you would have easier calculations.

Ferrous sulfate

Ferrous sulfate kdorfman Tue, 12/20/2011 - 15:16

Ferrous sulfate (FeSO4) 10 mM

MW (heptahydrate) = 278.01

MS 10x = 28mg/L = 0.1 mM - 100 µM

MS 1x = 0.01 mM = 10 µM

make 1,000x for M&S medium = 0.01 M = 10 mM

MW of FeSO4:
Concentration: M
to make: mL
add: g FeSO4

Keep refrigerated

Turns yellow. Probably oxidation. Yellow comes off on a micropore filter.

Fluorescein

Fluorescein kdorfman Mon, 10/29/2018 - 17:44

MW = 332.31

Excitation wavelength: 460 nm Emission wavelength: 515 nm

Absorption maxima at 493.5 and 460 nm

A = Ecl

Molar extinction coefficient of fluorescein at 485nm = 50358/mol.cm

so Absorbance 10^-5M = 0.50358

(1/100 of a 1mM solution =10^-5M)

1 mM stock in 10mM NaOH

MW of Fluorescein:
Concentration: M
to make: mL
add: g Fluorescein

fluorescein_info_sheet.pdf

Histidine HCl

Histidine HCl kdorfman Wed, 01/15/2020 - 21:41

100X Histidine-hydrochloride

8.56 g/L

Dissolve 2.14 g Histidine in 250 mL distilled water and filter sterilize.

Store in the dark at 4 ˚C.

IPTG

IPTG kdorfman Tue, 12/11/2018 - 20:47

1 M IPTG Stock Solution (isopropyl Beta-D-thioglucopyranoside, MW 238.3 g/mol)

2.383 g IPTG in 10 mL ddH2O.

Filter sterilize with 0.22 um membrane cartridge.

or buy 25 mL 1M stock solution Teknova 13431 (Fisher)

Store at -20C

Leucine

Leucine kdorfman Wed, 01/15/2020 - 21:43

10X Leucine

1.8 g/L

Dissolve 0.18 g Leucine in 100 mL distilled water and filter sterilize.

Store in the dark at 4 ˚C.

LiOAc-SDS

LiOAc-SDS kdorfman Mon, 07/31/2023 - 21:51

LiOAc-SDS for DNA extraction from yeast

To make 100 mL of

Mix 20 mL LiOAc

with 5 mL SDS

and bring to final volume

To make mL LiOAc-SDS solution
with mM LiOAC
and % SDS
Mix mL 1M LiOAc
and mL 20 % SDS

and bring to final volume

Lithium Acetate

Lithium Acetate kdorfman Wed, 01/15/2020 - 21:26

Lithium acetate dihydrate
C2H7LiO4
Fisher AA1341730
MW = 102.014

MW of LiOAc:
Concentration: M
to make: mL (start with about 90% of final volume)
add: g LiOAc

adjust pH to 7.5 with dilute acetic acid for Laney's cell & Molec lab

filter sterilize

store at room temp

Lithium Chloride

Lithium Chloride kdorfman Mon, 03/12/2012 - 14:28

LiCl 6M

MW = 42.39

MW of LiCl:
Concentration: M
to make: mL
add: g LiCl

Gets hot!!

MES

MES kdorfman Sun, 09/11/2016 - 17:47

MW = 195.2

150 mM stock solution for LPGM

MW of MES:
Concentration: M
to make: mL
add: g MES

Magnesium chloride

Magnesium chloride kdorfman Tue, 12/20/2011 - 15:05

Magnesium chloride (MgCl2) 1M

MW (hexahydrate) = 203.31

10.166 g/50 mL

20.33 g/100 mL

MW of MgCl2:
Concentration: M
to make: mL
add: g MgCl2

Magnesium sulfate

Magnesium sulfate kdorfman Tue, 12/20/2011 - 15:06

Magnesium sulfate (MgSO4) 1M

MW (heptahydrate) = 246.48

MW of MgSO4:
Concentration: M
to make: mL
add: g MgSO4

Manganese chloride

Manganese chloride kdorfman Thu, 01/19/2012 - 07:30

MnCl2 4H2O

M.W.197.9

5 mM

0.0495g/50mL

MW of MnCl2:
Concentration: M
to make: mL
add: g MnCl2

Manganese sulfate

Manganese sulfate kdorfman Tue, 12/20/2011 - 18:03

MnSO4

MW (monohydrate) = 169

store dry in dessicator

MS 10X micronutrients: 16.9 mg/L = 0.1 mM

so 1X = 0.01mM = 10 µM

1000X MS = 10 mM

MW of MnSo4:
Concentration: M
to make: mL
add: g MnSO4

Mannitol

Mannitol kdorfman Tue, 08/12/2014 - 17:45

MW = 182.172

Concentration: %
to make: mL
add: g mannitol

Potassium acetate

Potassium acetate kdorfman Tue, 12/20/2011 - 15:15

Potassium acetate (C2H3O2K)

Fisher BP364-500 $46.30

MW = 98.14

49 g in 100 mL H2O

MW of KOAc:
Concentration: M
to make: mL
add: g KOAc

Cloudy. Heat? Stir overnight? Lower the pH?

Can buy 5M solution: RICCA R5866500-1A ~$90 for 1L from Fisher

Potassium chloride

Potassium chloride kdorfman Tue, 12/20/2011 - 15:04

Potassium chloride (KCl)

MW = 74.56

MW of KCl:
Concentration: M
to make: mL
add: g KCl

Potassium ferricyanide

Potassium ferricyanide kdorfman Tue, 12/20/2011 - 15:13

Potassium ferricyanide (K3Fe(CN)6) 50 mM (= 0.05 M)

red salt

MW = 329.26

0.8232 g/50 mL

MW of Potassium ferricyanide:
Concentration: M
to make: mL
add: g Potassium ferricyanide

Store frozen

Potassium ferrocyanide

Potassium ferrocyanide kdorfman Tue, 12/20/2011 - 15:12

Potassium ferrocyanide (K4Fe(CN)6) 50 mM (= 0.05M)

yellow salt

MW (trihydride) = 422.41

MW of Potassium ferrocyanide:
Concentration: M
to make: mL
add: g Potassium ferrocyanide

Store frozen

Potassium hydroxide

Potassium hydroxide kdorfman Thu, 01/19/2012 - 16:33

KOH

MW = 56.11

MW of KOH:
Concentration: M
to make: mL
add: g KOH

Use for adjusting PIPES pH

Potassium nitrate

Potassium nitrate kdorfman Tue, 12/20/2011 - 15:18

Potassium nitrate (KNO3)

MW of KNO3:
Concentration: M
to make: mL
add: g KNO3

Store in refrigerator

Potassium phosphate dibasic

Potassium phosphate dibasic kdorfman Sat, 01/14/2012 - 05:06

K2HPO4

MW = 174.18

MW of K2HPO4:
Concentration: M
to make: mL
add: g K2HPO4

Potassium phosphate monobasic

Potassium phosphate monobasic kdorfman Tue, 12/20/2011 - 14:58

Potassium phosphate monobasic (KH2PO4)

MW of KH2PO4:
Concentration: M
to make: mL
add: g KH2PO4

for C. elegans medium:

pH to 6.0 with solid KOH

(~1.7 g/100 mL)

Sterilize (can be autoclaved)

SDS

SDS kdorfman Tue, 12/20/2011 - 14:40

SDS 20%

Sodium dodecanesulfate (=Sodium lauryl sulfate, NOT laureth)

CH3(CH2)11OSO3Na

Concentration: %
to make: mL
add: g SDS

DO NOT AUTOCLAVE - it (like all detergents) can boil over

Sodium acetate

Sodium acetate kdorfman Tue, 12/20/2011 - 15:08

Sodium acetate (NaOAc) 3M

CH3COONa

MW (trihydrate) = 136.08

MW of NaOAc:
Concentration: M
to make: mL
add: g NaOAc

pH 5.2

Sodium azide

Sodium azide kdorfman Tue, 12/27/2011 - 18:08

For PBS-Tw-Azide

NaN3

MW = 65

10% w/vol

Concentration: %
to make: mL
add: g NaN3

Sodium bicarbonate

Sodium bicarbonate kdorfman Tue, 12/20/2011 - 14:54

Sodium bicarbonate (CHNaO3) 0.5 M (=500 mM)

MW = 84.01

MW of Sodium bicarbonate:
Concentration: M
to make: mL
add: g sodium bicarbonate

Write the date on it. Probably only stable for 2 weeks.

Sodium chloride

Sodium chloride kdorfman Tue, 12/20/2011 - 14:36

Sodium Chloride (NaCl) 5 M

MW = 58.44

Concentration: M
to make: mL
add: g NaCl

Sodium ferric EDTA

Sodium ferric EDTA kdorfman Mon, 08/03/2015 - 18:43

NaFe(III)EDTA

FW = 367.05

50 mM

MW of NaFe(III)EDTA:
Concentration: M
to make: mL
add: g NaFe(III)EDTA

Sodium hydroxide

Sodium hydroxide kdorfman Mon, 10/29/2018 - 17:55

NaOH

MW = ~40

10mM = 0.01M = 0.4g/L

MW of NaOH:
Concentration: M
to make: mL
add: g NaOH

if you have g of NaOH pellets
which has a MW of g/mol
and you want to make M NaOH
dissolve in mL water

Sodium phosphate dibasic

Sodium phosphate dibasic kdorfman Tue, 12/20/2011 - 15:00

Use 0.2M for colorimetric exercise

Aliquote ~400 uL for a pair of students to each do the exercise

MW of Na2HPO4:
Concentration: M
to make: mL
add: g dry stuff

Use hot water

Sodium phosphate monobasic

Sodium phosphate monobasic kdorfman Tue, 12/20/2011 - 14:52

Sodium phosphate monobasic (NaH2PO4)

MW (monohydrate) = 137.99

Use 0.2M for colorimetric exercise

Aliquot ~400 uL - enough for 2 students to each do one exercise.

MW of NaH2PO4:
Concentration: M
to make: mL
add: g dry stuff

Sodium sulfate

Sodium sulfate kdorfman Thu, 03/01/2012 - 14:17

Na2SO4.10H2O

MW = 322.2

MW of Na2SO4:
Concentration: M
to make: mL
add: g Na2SO4

Spermidine

Spermidine kdorfman Fri, 05/31/2013 - 18:21

Spermidine:

  • neutralizes and stabilizes the negative charge on the DNA phosphate backbone
  • displaces other ions from DNA
  • stimulates restriction enzyme reactions
  • increases specificity and reproducibility of Taq-mediated PCR

Used in Gene & Genome

1 M stock

1 g spermidine (entire contents of bottle) in 6.9 mL water

MW = 145.25

Filter sterilize (0.22 µm filter, 10 mL syringe)

Freeze 200 uL aliquots

20 mM student solutions

1 g spermidine (entire contents of bottle) in 6.9 mL water

MW = 145.25 Make 20 mM solution for students by adding 800 uL to the 200 uL of 1M in the tube, then aliquot 10 uL for each student.

To make µL 20mM Spermidine
add: µL 1M spermidine
to: µL water

TBSTw

TBSTw kdorfman Thu, 11/17/2022 - 17:24

Tris Buffered Saline with Tween

  • Tris 20 mM
  • NaCl 100 mM
  • Tween 0.1%
  • pH 7.4
To make mL TBSTw
add: mL 1M Tris
add: mL 5M NaCl
add: mL 10% Tween

Tris

Tris kdorfman Tue, 12/20/2011 - 14:42

Tris 1M

Tris base MW = 121.14

MW of Tris:
Concentration: M
to make: mL
add: g Tris

pH to 7.5 or 8.0, depending on the application

Tris-HCl

Tris-HCl kdorfman Tue, 06/21/2022 - 17:26

MW 157.6

Tris-HCl 1M

MW of Tris-HCl:
Concentration: M
to make: mL
add: g dry Tris-HCl

Triton X

Triton X kdorfman Tue, 12/20/2011 - 15:17

Triton X 10%

MW = 646.86

1 g = ~1 mL

Concentration: %
to make: mL
start with: mL Triton X

Tryptophan

Tryptophan kdorfman Wed, 01/15/2020 - 21:42

50X Tryptophan

7.5 g/L

Dissolve 1.875 g Tryptophan in 250 mL of distilled water (reserve some water to rinse the sides of the beaker). Leave it on the stir plate for a while. It's powdery and resistant to going into solution.

Filter sterilize.

Store in the dark at 4 ˚C.

Tween 10%

Tween 10% kdorfman Tue, 12/27/2011 - 18:01

Tween 10%

for PBS-Tween-Azide

1 g = ~1 mL [density = 1.095 g/mL at 25 °C (from Sigma website)]

Concentration: %
to make: mL
start with: mL Tween

Do not autoclave

Sugars

Sugars kdorfman Mon, 09/12/2016 - 21:45

Glucose

Glucose kdorfman Mon, 09/12/2016 - 22:03

Molarity (MW = 198.17)

MW of glucose:
to make: mL
at a concentration of: M
start with: mL water
add: g glucose slowly, to already stirring water

Then bring to final volume. Filter sterilize

Percent

to make: mL
at a concentration of: percent
start with: mL water
add: g glucose slowly, to already stirring water

Then bring to final volume. Filter sterilize

Lactose

Lactose kdorfman Mon, 09/12/2016 - 22:07

MW = 342.3

MW of lactose:
Concentration: M
to make: mL
add: g lactose

heat water first, then tap in lactose while stirring.

Mannitol

Mannitol kdorfman Mon, 09/12/2016 - 22:08

MW = 182.17

MW of mannitol:
Concentration: M
to make: mL
add: g mannitol

Sucrose

Sucrose kdorfman Mon, 09/12/2016 - 22:02

MW = 342.3

MW of Sucrose:
Concentration: M
to make: mL
add: g sucrose

Solubility: 100 mg/mL in water at RT (= 10 g/100 mL)

Repairs

Repairs kdorfman Mon, 07/08/2013 - 18:15

Emergency building repairs 545-6401

Routine building repairs Service Request Form

Incubators & Refrigerators: Rene Cote (413) 534-5302

Biological Safety Cabinets: B&V Testing 800-851-9081

Autoclave Repairs: Ranger engineering (Get a $1000 PO to start) (508) 877-3166

Belimed Dishwashers: B-US_TechServicesAdministration b-us_techservicesadministration@belimed.com

P: +1 800 451 4118, Option 2

E: service.us@belimed.com

BELIMED INCORPORATED
2325 CHARLESTON REGIONAL PKWY
CHARLESTON, South Carolina 29492 United States

Manual for label printer in 362A

Restriction Enzymes

Restriction Enzymes kdorfman Fri, 08/26/2022 - 15:50

Updated Jan 2023

Diluent B

Enzyme volume 2023_01_05 CutSmart NEB1 NEB2 NEB3 notes
Aat2 ~10 100 10 50 50
Acc651 >100 25 10 75 100
Aci1 25 100 10 25 100 2 tubes - ordering more
AflII ~10 100 50 100 10
Age1 20 75 100 75 25
AlwN1 25 100 10 100 50
Apal1 >100 100 100 100 10
Asc1 20 100 10 10 10
Ava1 >100 100 10 100 25
Ava2 >100 100 50 75 10
BamHI-HF 100 100 100 50 10
BbsI-HF 1 100 10 10 10 "rCutsmart" 1 ul left - 2022 G&GA Maresca
BciVI <10 100 100 25 10
Bgl2 25 10 10 10 100 2 tubes
BmgB1 15 10 10 10 100
BsmAI 100 100 50 100 100 tiny amount
BsaI ~10 100 5 75 100
BsmB1 0 25 <10 50 100 use NEBuffer r3.1 - NONE- 2022 G&GA Maresca
BspD1 100 100 25 75 50
BspE1 25 10 10 10 100 from 2007
BsrB1 100 100 50 100 100
BsrD1 50
BsrG1 100 25 25 100 100
Bsu361 100
Cla1 100 100 10 50 50
Dra1 100 100 75 75 50
Eag1-HF 25 10 10 25 100
Eag1 25
Ear1 20 100 50 10 10
EcoR1 100 50 25 100 50 1 tube
EcoR1-HF over 500 50 25 100 50 3 Tubes
EcoRV-HF 100 100 25 100 100
HaeIII 100 100 50 100 25
Hha1 100
Hind3 500 50 25 100 50 2 tubes - tons of it
Hind3HF over 1000 100 10 100 10 3 tubes
HinP1I 200 100 100 100 100
Hlu1-HF 0 NONE
Hpa1 30 100 10 75 25 2 tubes
Hpa2 over 100 100 100 50 10
Kas1 100 100 50 100 50
KpnI-HF 300 100 100 25 10
Mbo1 25 100 75 100 100
Mlu1 100 25 10 50 100
Mlu1-HF 100 2 tubes
Mnl1 600 3 tubes
Msp1 250 100 75 100 50
Nae1 25 100 25 25 10
Nco1 100 100 100 100 100
Nco1-HF 50
Nde1 100 100 75 100 100
Nhe1-HF 10 100 100 25 10
Psha1 100
Pst1 25 50 75 75 100
Pvu1 100 10 10 25 100
Pvu1-HF 20
Pvu2 500 100 50 100 100
Rsa1 50 100 25 50 10
Sac1 25 100 100 50 10
Sal1 100
Sal1HF 75 10 10 10 100
Sau3A1 10 100 100 50 10
Sca1 50 NR NR NR 100
SexA1 50 100 100 75 50
SgrA1 100
Sma1 75 100 10 10 10
Spe1 (NEB) 25 100 75 100 25
Spe1GQ (Promega) 25
Sph1 25 100 100 100 50
SSpI (Fisher) 50 50 50 100 50 "Buffer G" recommended, P/N ER0771 Fisher
Stu1 25
Sty1 300 10 10 25 100
Sty1-HF 150 100 25 100 25
Xba1 225 100 10 100 75 3 Tubes
Xcm1 100 100 10 100 25
Xho1 100 100 75 100 100
Xma1 50
ExoSap-It under 1000 in "other enzymes"

2017 list

2016 list

Routine checks 2022

Routine checks 2022 kdorfman Mon, 02/07/2022 - 18:42

Safety Inspections

Safety Inspections kdorfman Tue, 05/29/2012 - 20:09

Bioraft safety reports

hazardous waste pickup (sign into CEMS)

emergency_eyewash_station-1.doc
haz-waste.doc

Suppliers

Suppliers margaret Thu, 10/13/2011 - 16:27

UMass BuyWays

mailto:dennis.glick@thermofisher.com

Fisher stockroom phone: 413-577-2600

Airgas

Airgas margaret Thu, 10/13/2011 - 17:18

Airgas East
1361 Union Street
W. Springfield, Ma.
Phone:800-649-1639
413-781-6550

Airgas

standing PO: A001284195
(updated summer 2022)

Account # 2516134

Liquid Nitrogen is in TC room 371

CO2 (CD75) (75lb cylinders) are in Prep Room 366A

They sell Radnor leak detection solution and dabber bottles but here is a recipe: (see Leak Detection Solution Recipe)

  • 946 ml water
  • 44.4 ml of dish detergent (Dawn)
  • 9.9 ml Glycerol*

The solution becomes more effective if you leave it overnight in an open container. In fact, the longer you leave it, the better the bubbles it produces.

*we've decided glycerin is the same thing as glycerol. It makes the bubbles last longer, which makes them easier to detect

Carolina Biological

Carolina Biological margaret Thu, 10/13/2011 - 16:48

Carolina Biological Supply Company

2700 York Rd.

Burlington, NC

27215-3398

Phone: 800-334-5551

Fax: 336-584-3399

http://www.carolina.com/

Fisher Scientific-Clonetech

Fisher Scientific-Clonetech margaret Thu, 10/13/2011 - 16:53

Fisher Scientific

2000 Park LN.

Pittsburgh, PA 15275 Clonetech

A Takara Bio Company

1290 Terra Bella Ave.

Mountain View, CA

94043

Phone:866-435-2566

Fax:800-424-1350

Contact Person: J. Fidago

http://www.clontech.com/

Krackler Scientific, Inc.

Krackler Scientific, Inc. margaret Thu, 10/13/2011 - 17:09

Krackler Scientific
PO Box 1849
Albany, NY
12201-1849

http://www.krackeler.com/

Qiagen

Qiagen margaret Fri, 10/14/2011 - 15:45

http://www.qiagen.com
Address: 27220 Turnberry Lane, Suite 200, Valencia, CA 91355
Hours:Normal business hours are 6:00 a.m. - 5:00 p.m. (PST) weekdays.
Telephone: Technical Service: 800-DNA-PREP (800-362-7737)
Customer Care: 800-426-8157
Fax:800-718-2056
Email:Customer Care (Ordering): customercare-us@qiagen.com
Literature Request: literature-us@qiagen.com

Rainin

Rainin margaret Thu, 10/13/2011 - 16:31

Rainin Road, Woburn Ma. 01888-4026

Phone: 781-935-3050

Order Line: 800-472-4646

http://www.manta.com/c/mmsfgny/rainin-instruments-co-inc

Part numbers

Part numbers margaret Thu, 10/13/2011 - 16:43

L-20 Pipet-Lite

L-200 Pipet-Lite

L-1000 Pipet-Lite

GPS-L1000 Spacesaver LTS 1000UL tip 768/8

GPS-L250 Spacesaver LTS 250UL tip 960/10

GPS-L10 Spacesaver LTS 20UL tip 960/10

GPR-L10 Empty Rack/Lid LTS 20UL RED 10/PKG

GPR-L250 Empty Rack/Lid LTS 250UL Green 10/PKG

GPR-L1000 Empty Rack/Lid LTS 1000UL Blue 8/PKG

Sigma-Aldrich

Sigma-Aldrich margaret Thu, 10/13/2011 - 17:13

Sgma-Aldrich
3050 Spruce Street
St. Louis, MO
63103
Phone:Customer Service 800-325-3010
Technical Service 800-325-5832
Fax:800-325-5052

http://www.sigmaaldrich.com/united-states.html

Thermo Fisher Scientific-Acros Organics

Thermo Fisher Scientific-Acros Organics margaret Fri, 10/14/2011 - 15:40

Thermo Fisher Scientific
New Jersey – US (8.00 AM to 6.00 PM Monday to Friday)
http://www.acros.com
General Information www.acros.com
Technical Support Tel : 1-800-227-6701
Email : chem.techinfo@thermofisher.com

Wahoo access

Wahoo access kdorfman Mon, 02/26/2018 - 18:34

From your Mac

From your Mac kdorfman Wed, 02/27/2019 - 19:12

On a networked Macintosh on campus

  • Open a finder window, and pull down the Go menu.
  • Choose “connect to server” and type “smb://wahoo.cns.umass.edu/bioimaging”.
  • Sign in with your BCRC username and password.

From anywhere else

Download and install FileZilla following the instructions on the OIT web-hosting support page (http://www.oit.umass.edu/support/web-hosting).

  • Open FileZilla.

  • Open Site Manager from the File menu, and enter the following:

    • Select Entry: New Site
    • Hostname: wahoo.cns.umass.edu
    • Port: 22
    • Protocol: SFTP – SSH File Transfer Protocol
    • Logon Type: Ask for password
    • User: your BCRC username
  • Click Connect.
  • Enter your BCRC password, and click OK
  • Note: The first time you log in, a one-time screen warning you about an unknown host key may appear. Check the box next to Always trust this host, add this key to the cache and click OK.
  • The remote site is something like this: /u1/home/bio/username (personalized with your username, of course).
  • Clear the Remote site box, and type this: /data/bioimaging
  • Enlarge the absurdly small window under the remote site bar and scroll to find your microscope folder. Move files between local and remote folders by drag-and-drop, or by right click and upload (local to remote) or download (remote to local).
    Voilá!

From your PC

From your PC kdorfman Wed, 02/27/2019 - 19:12

* Download and install <a href="http://www.umass.edu/it/support/web-hosting">WinSCP</a>

* Open Site Manager from the File menu, and enter:

 * Select Entry:  New Site
  * Hostname: wahoo.cns.umass.edu
 * port:  22
 * protocol:  SFTP-SSH File Transfer Protocol
 * Logon type:  ask for password
 * User: your BCRC username

* Click Connect

* Enter your bcrc password, and click OK

* If you get a warning about an unknown host key, choose "always trust this host" and add this key to the cache.

* The remote site is something like this: /u1/home/bio/username (personalized with your username, of course)

* Clear the Remote site box and type: /data/bioimaging

* Enlarge the absurdly small window under the remote site bar and scroll to find your microscope folder.  Move files between local and remote folders by drag-and-drop, or by right click and upload (local to remote) or download (remote to local)