Better Labs and Prep Rooms
Better Labs and Prep Rooms kdorfman Thu, 06/04/2009 - 17:01Here are the recipes for making solutions and doing stuff in the ISB.
2022 Summer
2022 Summer kdorfman Fri, 04/29/2022 - 16:17Get Mary authenticated on WahooSet up "permanent" groups for orders and cell lines spreadsheetsEmpty 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 mixersLadders / DNA Standards Inventory (1 Kb for QBoC)Defrost freezer 266ADefrost freezer 362AClean microscopes 264*Clean microscopes 364- Sharpen forceps
Refill Vaseline syringes- organize mini-drawers
Pipette rodeoPour plates
100 LB25060 YED150 MV70 MVADE60 YeKAc
Inventory and replenish
order Aci1, Mlu1, XBa1, 1 Kb ladder from NEB #B7025major equipment (with purchasing)mini-prepstipstubessterile toothpickssterile 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 repairClear out 366A for Animal ScienceMove -80 from Lederle to ISBMove into new -80- Inventory E. coli
Move LN2 to371
Chemical delivery address
Chemical delivery address kdorfman Tue, 01/07/2020 - 21:47University 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 - 18:39username: .\admin
pw :
To find the computer's name:
rt click start menu
run
cmd
ipconfig /all
Courses
Courses kdorfman Thu, 01/11/2024 - 18:27Courses taught in ISB
Bio 499CB (Loomis Cancer)
Bio 499CB (Loomis Cancer) kdorfman Thu, 09/08/2022 - 20:59Kari 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
Marine Biology
Marine Biology kdorfman Mon, 01/15/2024 - 17:32Growth chamber spawning program
Growth chamber spawning program kdorfman Fri, 05/17/2024 - 19:17Basic Parameters:
Light from 9 pm Sunday to 1 pm Monday, otherwise dark
Warm from 9 pm Sunday til 9 am Monday, otherwise cool
Day | time | temp | light level |
---|---|---|---|
M | 13:00 | 18 | 0 |
Sun | 21:00 | 25 | 3 |
M | 9:00 | 18 | 3 |
M | 13:00 | 18 | 0 |
Schedule 3 programs, like this:
Program 1, repeat 6 times:
Day | time | temp | light level |
---|---|---|---|
M | 00:00 | 18 | 0 |
Program 2 (spawning), do once, following 6x Program 1
Day | time | temp | light level |
---|---|---|---|
Sun | 00:00 | 18 | 0 |
21:00 | 251 | 3 |
Program 3, do once
Day | time | temp | light level |
---|---|---|---|
M | 00:00 | 25 | 3 |
9:00 | 18 | 3 | |
13:00 | 18 | 0 |
Return to Program 1
-
There is a temperature spike when the light comes on, so leave the temp setting at 18 for an hour (until 22:00), then raise it to 25. ↩︎
Sea Water for 424
Sea Water for 424 kdorfman Mon, 01/15/2024 - 18:142 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
F/2 Medium for algae (424)
F/2 Medium for algae (424) kdorfman Mon, 01/15/2024 - 18:165 liters of f/2 medium for algae.
L1 Medium Kit from Bigelow NCMA contains the nitrate, phosphate, trace minerals, and vitamins. Add the antibiotics from prep room freezer stocks.
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.
Plus ASW to final volume
Filter sterilize
Trace minerals for F/2
Trace minerals for F/2 kdorfman Mon, 06/24/2024 - 15:46Buy L1 Medium Kit from Bigelow NCMA
Replacement trace minerals solution
Component | Concentration in final medium |
---|---|
Na2EDTA · 2H2O | 1.17 x 10-5 M |
FeCl3 · 6H2O | 1.17 x 10-5 M |
MnCl2·4 H2O | 9.00 x 10-7 M |
ZnSO4 · 7H2O | 8.00 x 10-8 M |
CoCl2 · 6H2O | 5.00 x 10-8 M |
CuSO4 · 5H2O | 1.00 x 10-8 M |
Na2MoO4 · 2H2O | 8.22 x 10-8 M |
H2SeO3 | 1.00 x 10-8 M |
NiSO4 | 1.00 x 10-8 M |
Na3VO4 | 1.00 x 10-8 M |
K2CrO4 | 1.00 x 10-8 M |
Vitamins for F/2
Vitamins for F/2 kdorfman Mon, 06/24/2024 - 15:51Buy L1 Medium Kit from Bigelow NCMA
Replacement Vitamin Stock Solution
Component | Concentration in Final Medium |
---|---|
thiamine · HCl (vit. B1) | 2.96 x 10-7 M |
biotin (vit. H) | 2.05 x 10-9 M |
cyanocobalamin (vit. B12) | 3.69 x 10-10 M |
Perfect Plant 2024
Perfect Plant 2024 kdorfman Wed, 01/10/2024 - 21:51Bio 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 |
2024/02/07
2024/02/07 kdorfman Thu, 01/11/2024 - 18:47Perfect 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 - 19:01Perfect 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 - 19:06Perfect 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 - 21:37Individual Plant Day 1
2024/03/27
2024/03/27 kdorfman Sun, 01/28/2024 - 15:54Display Easels
Display Easels kdorfman Mon, 05/12/2014 - 15:17Looking 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
Door Access
Door Access kdorfman Fri, 06/07/2024 - 17:01prefers to communicate with just one Biology Department member (currently Kate Dorfman)
In order to grant swipe access, DoorAccess needs names and SPIRE numbers.
Undergrads must be trained in the use of the equipment in the lab they can access.
Electronics and Equipment
Electronics and Equipment kdorfman Wed, 10/19/2011 - 16:27Computers, servers, etc.
Computers, servers, etc. kdorfman Wed, 08/19/2009 - 16:55The 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 - 15:35Go 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 @$74.95
Screen Capture
Screen Capture rootlet Fri, 04/02/2010 - 14:46You can capture still images of videos of anything on the computer screens that Biology supports.
Capture Stills
To capture still images, you can use some magic keystrokes:
Capture whole screen: Command + Shift + 3
Capture region: Command + Shift + 4 to turn cursor to cross-hairs, then select region.
There is also an application in the Utilities folder called "Grab" that will let you set some options or get images just of particular windows.
Capture Video
To capture video, you need to use the command-line. Videos of the entire screen are very large. You might want to use the System Preferences to set the screen resolution lower before starting to capture video. We're using the vnc2flv project. Start up Terminal (Also in the Utilities folder, but there a shortcut in the Dock). You can use two commands to start recording video. With any luck, it will be as simple as this:
First, go to the Desktop to save your work there: delfeno:~ sbrewer$ 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 - 15:41Using 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 - 20:57Autoclaves:
- 261,
- 361
- Consolidated Sterilizer
- Model SR-24C-PB
- SR 071608
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
Convection Oven
Convection Oven margaret Fri, 10/14/2011 - 16:01Detergent for Dishwasher
Detergent for Dishwasher margaret Wed, 10/12/2011 - 17:09Order from Fisher Scientific: Catalog Number-04319B Detergent Liquid Nalgene L900
http://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&pr…
Plant growth chambers
Plant growth chambers kdorfman Tue, 10/27/2020 - 16:45ISB 373
Conviron Adaptis A1000
2 set up for Arabidopsis:
089150
089149
1 set up for tissue culture 089136
HOBO Data Logger
HOBO Data Logger kdorfman Fri, 05/24/2024 - 16:07Motic Camera & Software
Motic Camera & Software margaret Wed, 10/12/2011 - 16:03Plug 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
2021 (June) Motic inventory
2021 (June) Motic inventory kdorfman Tue, 06/08/2021 - 20:19MP | part # | live | dead | notes |
---|---|---|---|---|
5.0 | 2500 | 1 | very slow on dissecting trinoc in 360 | |
2.0 | 2300 | 9 | 4 | (9 including 1 on trinoc in 360) |
1.3 | 1000 | 10 |
Projectors
Projectors kdorfman Wed, 06/17/2009 - 14:50There 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 - 19:34Room | 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 |
Jenway Genova Spectrophotometer
Jenway Genova Spectrophotometer kdorfman Tue, 09/12/2023 - 14:06Several in whiteboard cupboards in ISB 368
Manual can be found at Manualzz.com
Nikon E200
Nikon E200 kdorfman Thu, 08/28/2014 - 16:46To clean and troubleshoot:
make sure lamp housing is all the way in
make sure swing-out condenser is all the way in or out.
check underside of ocular head for fingerprints
pointer goes under the viewfield ring
See manual here: http://www.micropticsl.com/wp-content/uploads/2013/09/nikon_e200_manual…
Optima Plate Reader
Optima Plate Reader kdorfman Tue, 01/17/2017 - 19:12Link 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 |
Plate Reader Programs
Plate Reader Programs kdorfman Thu, 10/13/2022 - 15:20Look 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
-
Lab 3.1 (2022) counting bacteria: std curve OD vs population density ↩︎
-
Lab 4.2 (2022) lac operon shake cells, then read OD for overnight growth curves – part of script “Grow&Glow” ↩︎
-
Lab 4.2 (2022) lac operon after BugOD Shake,read FL for overnight growth curves– part of script “Grow&Glow” ↩︎
-
Lab 1.3 serial dilution; absorbance of fluorescein; std curve ↩︎
-
Lab 1.3 serial dilution; fluorescence of fluorescein; std curve ↩︎
Script for lac operon lab
Script for lac operon lab kdorfman Mon, 10/24/2022 - 15:18Script 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 - 20:28OptixCam
(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)
Thermocyclers
Thermocyclers kdorfman Tue, 06/27/2023 - 14:44Zeiss scopes
Zeiss scopes kdorfman Tue, 01/28/2014 - 16:02Phase scopes from Bio.
Borrowed for AnSci F13, then for QSB S14
Equipment Lending
Equipment Lending kdorfman Mon, 06/19/2017 - 19:27date 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 - 19:38Handouts
Handouts kdorfman Thu, 05/02/2019 - 18:51CoolLED handout to sit next to microscope
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
Stage Micrometer images
Stage Micrometer images kdorfman Mon, 02/26/2024 - 18:49Magnification | pixels per micron |
---|---|
2X | 0.4305 |
4X | 0.858 |
10X | 2.13 |
20X | 4.273 |
40X | 8.687 |
100X | 21.403 |
ISB Rooms
ISB Rooms kdorfman Thu, 01/19/2012 - 22:37Classrooms
Classrooms kdorfman Thu, 01/19/2012 - 22:38Conference rooms
Conference rooms kdorfman Thu, 01/19/2012 - 22:38Mail room: 147
Keys
Keys kdorfman Wed, 09/04/2019 - 15:04IC-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 - 22:37255 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 - 19:402020 Spring
2020 Spring kdorfman Tue, 12/20/2011 - 20:33Dept | # | 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 - 19:52Class | 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 - 19:30Class | 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 - 21:48Room | 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 - 18:55Dept | 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 - 15:14Calendars
360 S2024
360 S2024 kdorfman Tue, 11/21/2023 - 15:15264 S2024
264 S2024 kdorfman Tue, 11/21/2023 - 15:16Dept | 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 - 15:16Dept | # | 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 - 15:17Dept | # | 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 - 15:17Dept | # | 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 - 14:15These 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.
0.5 mL microtube labels
0.5 mL microtube labels kdorfman Wed, 06/17/2009 - 14:35Use 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!
1.5 mL microtube labels
1.5 mL microtube labels kdorfman Wed, 06/26/2013 - 20:37Brother P-touch 55
Brother P-touch 55 kdorfman Fri, 08/25/2017 - 16:50Drawer labels
Drawer labels kdorfman Wed, 06/17/2009 - 14:22Use 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.
Slide Labels
Slide Labels kdorfman Wed, 08/19/2009 - 17:15These 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.
Spot Labels
Spot Labels kdorfman Wed, 01/27/2010 - 14:33Fisher Scientific
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
- Log in to post comments
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
dH2O labels
dH2O labels kdorfman Thu, 03/03/2016 - 14:03Avery 5160
Misc supplies
Misc supplies kdorfman Mon, 12/16/2013 - 19:11Sterile 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 - 14:39Disposable 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 - 18:42dates | 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 - 17:53Billing 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
- Enter any OneCard orders Manually
- 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 - 16:13Create 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 - 14:152022 Eureka
2022 Eureka kdorfman Thu, 07/14/2022 - 19:27color 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 - 13:47Staining 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 - 15:482016 Bio Bootcamp
2016 Bio Bootcamp kdorfman Wed, 06/29/2016 - 17:192017 bio boot camp
2017 bio boot camp kdorfman Tue, 06/20/2017 - 18:0224 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
2018 Bio Boot Camp
2018 Bio Boot Camp kdorfman Mon, 07/02/2018 - 22:432022 Bio Boot Camp
2022 Bio Boot Camp kdorfman Wed, 06/22/2022 - 18:20FIRST DAY
Pipetting Exercise
Weigh 20 uL, 200 uL, 1000 uL to check pipets
Enter data into pipet spreadsheet
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
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
- 1 1% agarose gel per pair
- SYBR Safe 1 uL/10 mL agarose
- small gel (50 mL)
- ? small tooth comb, 2 per gel
- 1X TAE
- loading dye
- DNA standard
BLAST
Use primer sequences?
Analyze Gel
- check bands in blue light
- take cell phone photo
- send to Rolf with documentation
-
45 sec is better for mcherry; can use 45 sec to 1 min for GFP. EGFP and mcherry amplicons are ~500 BP ↩︎
Master mix
Master mix kdorfman Mon, 06/12/2023 - 15:05Master Mix, multiple primers
Master Mix, multiple primers kdorfman Mon, 07/01/2024 - 22:10NOT SURE WHY THIS DOESN"T CALCULATE
2023 Boot Camp
2023 Boot Camp kdorfman Wed, 06/21/2023 - 18:53- streamlined pipet exercise
- Genotyping of Rolf's fish
Zebrafish finclip genotyping
Zebrafish finclip genotyping kdorfman Wed, 06/21/2023 - 18:56Copied 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.
Set up PCR reactions
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
- 1 1% agarose gel per pair
- SYBR Safe 1 uL/10 mL agarose
- small gel (50 mL)
- ? small tooth comb, 2 per gel
- 1X TAE
- loading dye
- DNA standard
BLAST
Use primer sequences?
Analyze Gel
- check bands in blue light
- take cell phone photo
- send to Rolf with documentation
-
45 sec is better for mcherry; can use 45 sec to 1 min for GFP. EGFP and mcherry amplicons are ~500 BP ↩︎
2024 Bio Bootcamp
2024 Bio Bootcamp kdorfman Wed, 05/29/2024 - 18:12Chestnut Middle School 2015
Chestnut Middle School 2015 kdorfman Tue, 06/02/2015 - 17:19Eureka 2015
Eureka 2015 kdorfman Thu, 07/30/2015 - 19:13MassBio
MassBio kdorfman Sat, 12/05/2015 - 14:16DIY
DIY kdorfman Sat, 12/05/2015 - 14:20Equipment
Equipment kdorfman Sat, 12/05/2015 - 14:23Paper Activities
Paper Activities kdorfman Sat, 12/05/2015 - 14:25Middle school 2016
Middle school 2016 kdorfman Tue, 06/14/2016 - 15:37Science Quest 2014
Science Quest 2014 kdorfman Tue, 04/08/2014 - 18:24Participant | 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? |
Summer Genetics
Summer Genetics kdorfman Wed, 07/11/2018 - 22:25Pipettes and tips
Pipettes and tips kdorfman Thu, 06/18/2009 - 16:04Classroom pipettes
Classroom pipettes kdorfman Thu, 01/03/2013 - 17:41The 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)
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 - 18:16Mix 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 |
Eppendorf Repeater
Eppendorf Repeater kdorfman Mon, 09/04/2023 - 20:17Dial | # 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 |
Prep Room Pipettes
Prep Room Pipettes kdorfman Thu, 01/03/2013 - 18:51Rainin
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
Get prices on tips from Krackeler
Protocols
Protocols kdorfman Tue, 06/16/2009 - 20:02How to do stuff
Sterilization
Sterilization kdorfman Tue, 06/16/2009 - 17:50Sterilize 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.
Autoclave
Autoclave kdorfman Tue, 06/16/2009 - 20:25Always 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
Minutes = 8.3853 * volume^0.2449
Monthly Maintenance
Monthly Maintenance kdorfman Fri, 09/09/2016 - 15:45Turn 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 - 13:45Container | 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 - 20:35There 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)
Drierite Regeneration
Drierite Regeneration margaret Thu, 10/06/2011 - 18:34For 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.
EtBr removal
EtBr removal kdorfman Tue, 04/03/2012 - 22:49Destaining bags from Amresco
http://www.amresco-inc.com/DESTAINING-BAGS-E732.cmsx
Fisher D300025 StainEx Destaining Bag, 25 bags
Or Green Bag Kit
Fisher NC9633024 $142.14
Incineration
Incineration kdorfman Thu, 12/29/2011 - 19:46Boxes and bags available in first floor cold room
Call EH&S for pick up 545-2682
or go here: https://cems.unh.edu/umass/CEMS/RequestRemoval
Mini-prep
Mini-prep kdorfman Thu, 09/15/2022 - 15:33Keep stocks of 2 mini-prep kits:
Qiagen
Zymo Classic D4015
Qiagen
Qiagen kdorfman Thu, 09/15/2022 - 19:53Jeff's CMBL miniprep
Zymo miniprep classic
Zymo miniprep classic kdorfman Thu, 09/15/2022 - 19:53For 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 (or spun down pellet of ON culture)
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
-
All the ingredients are sold separately, so it's OK to give students some extra to allow for pipetting errors. ↩︎
Peroxide testing
Peroxide testing kdorfman Tue, 10/09/2018 - 20:46Preparation
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.
Weekly Inspections
Weekly Inspections kdorfman Tue, 10/11/2011 - 16:47Biohazard Waste
Biohazard Waste kdorfman Tue, 10/11/2011 - 16:50Check 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.
Facility Inspection Check List
Facility Inspection Check List margaret Tue, 11/08/2011 - 17:19Recipes
Recipes kdorfman Tue, 06/16/2009 - 20:04Antibodies
Antibodies kdorfman Tue, 10/02/2018 - 16:21For 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 - 16:215-mC (rabbit)
5-mC (rabbit) kdorfman Fri, 11/05/2021 - 13:445-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 - 18:50stock 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 - 19:04Dilute 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
Alkaline phosphatase (mouse)
Alkaline phosphatase (mouse) kdorfman Tue, 10/02/2018 - 18:51stock 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 - 17:46Binds 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 - 18:59stock 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 - 19:00mouse 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 (rabbit)
Connexin (rabbit) kdorfman Tue, 10/02/2018 - 19:07Freezer 266A
Anti-connexin-32 (265-279)
Dilute 1:600
Anti-Connexin-32 (106-124)
Dilute 1:400
Connexin (rat)
Connexin (rat) kdorfman Tue, 10/02/2018 - 19:17stock 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 - 13:39D63A6 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 - 17:111 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 - 16:23Novus Biologicals, recombinant human Hec1, monocolonal
fix in methanol
dilute 1:200
Histone H3 phospho S10
Histone H3 phospho S10 kdorfman Fri, 11/04/2022 - 20:46FINISH WORKING ON THIS!
Rabbit polyclonal
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 - 19:18rat 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 - 16:22IgG
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
- in 4% paraformaldehyde 15 min for endosomes
- in cold methanol for Golgi, 0.5% saponin 5 min
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
LAP2 (mouse)
LAP2 (mouse) kdorfman Tue, 10/02/2018 - 18:48Stains nuclear envelope
- fix in cold methanol
- 1:100
- BD transduction
- Mouse IgG
Lamin A/C
Lamin A/C kdorfman Fri, 11/04/2022 - 20:36mouse 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 - 16:30Rabbit 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 - 19:01mouse 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
Laminin (rabbit)
Laminin (rabbit) kdorfman Tue, 10/02/2018 - 19:08https://www.sigmaaldrich.com/catalog/product/sigma/l9393?lang=en®ion…
3 vials as of Spring 2018
dilute 1:25
266A freezer
Mucin (mouse)
Mucin (mouse) kdorfman Tue, 10/02/2018 - 19:03Monoclonal Anti-Mucin Gastric antibody produced in mouse
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
Myosin (mouse)
Myosin (mouse) kdorfman Tue, 10/02/2018 - 18:46mouse 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 - 19:14Neurofilament (rabbit)
Neurofilament (rabbit) kdorfman Tue, 10/02/2018 - 19:16Nuclear pore complex
Nuclear pore complex kdorfman Wed, 10/18/2023 - 19:16Anti-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
PKC (rabbit)
PKC (rabbit) kdorfman Tue, 10/02/2018 - 20:44PKC 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.
Phospho H3 (rabbit)
Phospho H3 (rabbit) kdorfman Mon, 08/19/2024 - 17:55For showing mitotic cells
Histone H3 phosphorylation on serine-10 is specific to mitosis and phosphorylated histone H3 (PHH3) proliferation markers (as counts defined per area or as indices defined per cell numbers) are increasingly being used to evaluate proliferation in various tumors.
Dilute 1:2000
Synaptic vesicle (mouse)
Synaptic vesicle (mouse) kdorfman Tue, 10/02/2018 - 18:57Synaptic 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 - 18:58DSHB TI-4-s
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 - 16:23Accurate 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 - 17:17binds 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
-
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 ↩︎
Vinculin (mouse)
Vinculin (mouse) kdorfman Tue, 10/02/2018 - 18:44Membrane-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 - 16:24large 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 - 19:19Goat anti-mouse IgM TRITC
Goat anti-mouse IgM TRITC kdorfman Tue, 10/02/2018 - 19:26Fisher OB102102 (Southern BioTech 1021-02)
Goat Anti-Mouse IgM (micron chain specific)-TRITC
1 mg/mL
1:100 - 1:400
1 µL aliquots. Add 100 - 400 µL 1% BSA
Use 50 µL per coverslip
Goat anti-mouse 488
Goat anti-mouse 488 kdorfman Tue, 10/02/2018 - 20:37Ex: 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
Goat anti-mouse TRITC
Goat anti-mouse TRITC kdorfman Tue, 10/02/2018 - 19:23Anti-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 546
Goat anti-rabbit 546 kdorfman Tue, 10/02/2018 - 20:39Stock concentration = 2 mg/mL
Working concentration = 4 ug/mL
Em = 575 nm
Ex = 560 nm
ALL OUT as of Spring 2018
Goat anti-rabbit FITC
Goat anti-rabbit FITC kdorfman Tue, 10/02/2018 - 20:42Goat anti-rabbit red
Goat anti-rabbit red kdorfman Tue, 10/02/2018 - 19:25Jackson 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 - 19:24Sigma 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 - 19:31From 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 - 17:51Here are some commonly made buffers for biochemistry and molecular biology
Citric acid buffer
Citric acid buffer kdorfman Tue, 11/16/2021 - 20:03Citric 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 - 17:40DNA Extraction Buffer for Gene & Genome
- 100 mM NaCl
- 50 mM Tris, pH8
- 25 mM EDTA
- 1% SDS
Make from stock solutions
Diluent B
Diluent B kdorfman Mon, 09/26/2022 - 20:21Dilution 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
Edwards Extraction Buffer
Edwards Extraction Buffer kdorfman Tue, 03/22/2022 - 13:20For quickie DNA extraction from Arabidopsis
- 200 mM Tris pH 7.5
- 250 mM NaCl
- 25 mM EDTA
- 0.5% SDS
HBS (Ca-)
HBS (Ca-) kdorfman Tue, 12/27/2011 - 17:53Calcium-free HBS
Make from dry ingredients:
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 - 20:32Ingredient1 | 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 |
-
Concentrations from Lab 9, Bioimaging 2008, Dave Gross ↩︎
10X HBS (dry)
10X HBS (dry) kdorfman Fri, 08/09/2013 - 18:5510X HBS/Ca/Mg from dry ingredients
pH 7.3 with NaOH
Filter sterilize
10X HBS (stocks)
10X HBS (stocks) kdorfman Fri, 08/09/2013 - 18:561X HBS (stocks)
1X HBS (stocks) kdorfman Fri, 08/09/2013 - 20:22pH 7.3 with NaOH
Filter sterilize
HBSS
HBSS kdorfman Fri, 04/03/2020 - 14:57Hank's Balanced Salt Solution
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 - 14:16HEPES 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 - 15:00For 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
Alkaline Lysis Reagent
Alkaline Lysis Reagent kdorfman Tue, 06/21/2022 - 15:07Alkaline 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
pH =~12
Neutralization Buffer
Neutralization Buffer kdorfman Tue, 06/21/2022 - 15:09PBS
PBS kdorfman Tue, 06/16/2009 - 17:52Make 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 |
pH to 7.3 with NaOH
sterilize
10X PBS from dry ingredients (Carrie's recipe)
pH to 7.3 with NaOH
sterilize
from stock solutions
from stock solutions kdorfman Wed, 11/13/2013 - 18:1310X 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 |
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
PBS-Tw-Azide
PBS-Tw-Azide kdorfman Tue, 12/27/2011 - 18:34PBS with:
- 0.1% Tween 20
- 0.2 g/L Na Azide (=0.02 %)
per Liter:
- 100 mL 10x PBS
- 10 mL 10% Tween 20
- 2 mL 10% Na azide
PBX (for fish)
PBX (for fish) kdorfman Mon, 03/23/2020 - 13:570.5% Triton X in PBS
Plus water to final volume
PBX/BSA
PBX/BSA kdorfman Tue, 11/10/2020 - 18:44PBX with 3% BSA for fish
PIPES
PIPES kdorfman Thu, 01/19/2012 - 16:430.5 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 - 04:581 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
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 - 19:20To 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 - 19:53From 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 - 17:54Tris 10 mM, EDTA 1 mM, pH 8
Make from stock solutions
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 - 18:25Tris 10 mM, EDTA 5 mM, pH 8
Make from stock solutions
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 - 17:52Tris-Acetate-EDTA buffer for agarose gel electrophoresis
1X TAE
1X TAE kdorfman Tue, 06/16/2009 - 19:02plus water to final volume
50X TAE stock
50X TAE stock kdorfman Tue, 06/16/2009 - 19:01Buy from Fisher TAE 50x, pH 8.3, 4L, part #BP13324
from Maniotis
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.
TBE 10X stock
TBE 10X stock kdorfman Thu, 08/29/2024 - 22:14BE Buffer 10x Stock Recipe:
- 108 g tris base.
- 55 g boric acid.
- 900 ml double-distilled H2O.
- 40 ml 0.5 M EDTA solution (pH 8.0)
Taq dilution buffer
Taq dilution buffer kdorfman Fri, 05/31/2013 - 17:21To dilute Takara Ex-Taq
Store in freezer. Won’t freeze solid. Should keep forever.
50 μL Tween 20
50 μL Nonidet P-40 (anionic detergent = NP-40)
10 µL 1M DTT
1 mL KCl (1M)
200 μL Tris-Cl pH 8 (1M)
2 μL 0.5M EDTA pH 8
5 mL glycerol
4.2 mL ddH2O
bring to final volume, filter sterilize in BSC, freeze
Culture Media
Culture Media kdorfman Fri, 04/27/2018 - 15:16Bacterial media
Bacterial media kdorfman Thu, 01/17/2013 - 18:53Freezing bacteria
Freezing bacteria kdorfman Sat, 12/05/2015 - 18:44To 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 - 17:54LB broth from mix
LB broth from mix kdorfman Tue, 05/28/2013 - 17:48LB Broth Miller Luria-Bertani
Fisher DF0446-17-3 500 g $40.61.
25 g/L
LB from scratch
LB from scratch kdorfman Fri, 09/25/2020 - 17:38LB (Miller) from scratch
LB agar
LB agar kdorfman Thu, 01/17/2013 - 18:55LB agar granules
LB agar granules kdorfman Tue, 06/25/2019 - 20:01Make with granulated LB (Miller) agar, Fisher BP9724
LB agar powder
LB agar powder kdorfman Tue, 06/25/2019 - 20:00Make with LB (Miller) Agar powder, Difco 244510
LB broth + agar
LB broth + agar kdorfman Tue, 06/25/2019 - 20:0025 g LB + 15 g agar /liter
LB antibiotic
LB antibiotic kdorfman Wed, 10/01/2014 - 16:34Table of antibiotic stability in poured agar
line | antibiotic |
---|---|
black | Carbenicillin (ampicillin) |
green | IPTG |
LB-sugars
LB-sugars kdorfman Wed, 10/01/2014 - 16:19LB + sugars for Lac-operon work
sugar (or analog) | MW |
---|---|
mono saccharide | 180.2 |
disaccharide | 342.3 |
IPTG | 238.3 |
glucose, galactose, or fructose
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.
lactose, maltose, sucrose
IPTG
Filter sterilize
Add 50 µg/mL Kanamycin as indicated here.
MacConkey agar
MacConkey agar kdorfman Thu, 01/17/2013 - 19:41MacConkey 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)
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 - 16:00If you accidentally buy MacConkey without
- bile salts (1.5 g/L)
- Crystal violet (0.001g/L)
- NaCl (5 g/L)
Min med for GFP cells
Min med for GFP cells kdorfman Tue, 10/23/2018 - 15:53From https://wahoo.cns.umass.edu/node/857
For SfGFP cells, try M9 + 0.2% glycerol + 0.01 mM FeSO4
Mueller Hinton agar
Mueller Hinton agar kdorfman Thu, 01/17/2013 - 20:0590922 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
P-Glo Bacterial media
P-Glo Bacterial media kdorfman Mon, 08/26/2019 - 22:26Make 60 mm plates
plain LB plates
LB/Amp (or Carb) plates (1 black stripe)
LB/Amp/Ara plates (1 black, 1 red stripe)
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.
LB-Amp-Ara
LB-Amp-Ara kdorfman Mon, 08/26/2019 - 22:27LB 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 - 17:34For motility strains
Cell culture media
Cell culture media kdorfman Tue, 10/11/2011 - 19:24FBS aliquots
FBS aliquots kdorfman Tue, 10/11/2011 - 20:15FBS 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 - 20:21Anti-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 - 20:00from Kathleen Arcaro
bring to final volume, filter sterilize in BSC, refrigerate
DMEM
DMEM kdorfman Tue, 10/11/2011 - 19:30Medium for 3t3 fibroblasts and B16 cells
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 - 19:49for all LLCPk cell lines
Thaw FBS and anti-anti first!
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 - 20:42Medium with 15% DMSO and 20% serum to protect cells in liquid nitrogen.
F10 Hams for freezing (initial serum concentration = 0.075)
DMEM for freezing (initial serum concentration = 0.1)
Filter sterilize
McCoy's 5A
McCoy's 5A kdorfman Tue, 09/19/2023 - 15:19McCoy'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
Non-CO2 Media
Non-CO2 Media kdorfman Tue, 12/27/2011 - 18:37Try 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 - 15:07Non-CO2 serum-free medium
For working with live cells at the microscope
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 - 20:00Non-CO2 Medium
For working with live cells at the microscope, when serum is needed for normal division.
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 - 18:58Try 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 - 21:48Use 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
0.1% Methylene Blue
0.1% Methylene Blue kdorfman Thu, 03/19/2020 - 14:531 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 - 14:54E2A (100x)
E2A (100x) kdorfman Thu, 03/19/2020 - 14:51FOLLOW 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
E2A Buffer Mix
E2A Buffer Mix kdorfman Thu, 03/19/2020 - 14:52E2B 500X
E2B 500X kdorfman Thu, 03/19/2020 - 14:52E2C 500X
E2C 500X kdorfman Thu, 03/19/2020 - 14:53Naegleria medium
Naegleria medium kdorfman Wed, 01/17/2018 - 14:34Medium 21
Medium 21 kdorfman Wed, 12/15/2021 - 21:44Mark 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 - 21:19Add 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 - 21:09If 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 - 14:34If 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 - 14:35DIW (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.
Nematostella
Nematostella kdorfman Sat, 06/22/2024 - 18:18Anemones with Symbiodinium (symbiotic photosynthetic dynoflagellates) for Marine Bio
Nematostella culture instructions powerpoint
Culture in 12 - 15 psu (practical salinity units = g/L) ASW (artificial sea water), made with Instant Ocean Reef Crystals
Plant growth media
Plant growth media kdorfman Thu, 05/23/2013 - 16:0210 g agar per L
1/2 MS low Iron
1/2 MS low Iron kdorfman Tue, 02/15/2022 - 15:001/2 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
Fern Culture Media
Fern Culture Media kdorfman Thu, 01/11/2024 - 19:16Pre-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
Fern Medium from scratch
Fern Medium from scratch kdorfman Fri, 01/12/2024 - 21:18To 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 - 21:24200X fern micronutrients
200X fern micronutrients kdorfman Fri, 01/12/2024 - 21:26Fern medium from powder
Fern medium from powder kdorfman Wed, 01/24/2024 - 14:20Mix 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 - 20:24MS high salt
MS high salt kdorfman Mon, 02/03/2014 - 15:33150 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 - 17:46Murashige & 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
MS low iron
MS low iron kdorfman Thu, 05/23/2013 - 17:40Murashige & 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
MS medium
MS medium kdorfman Thu, 05/23/2013 - 16:021/2 MS
1/2 MS kdorfman Wed, 12/22/2021 - 20:25*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*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 - 18:18Saburaud dextrose agar
Saburaud dextrose agar kdorfman Fri, 04/14/2023 - 18:19Before 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
Symbiodinium
Symbiodinium kdorfman Sat, 06/22/2024 - 18:07Algae for Nematostella anemones in marine bio
culture instructions powerpoint
Subculture monthly 1 mL of old culture into 50 mL sterile f/2 medium in 125 mL erlenmeyer flask
VTM
VTM kdorfman Fri, 04/03/2020 - 14:42Viral 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
- HBSS 1X with calcium and magnesium, without phenol red
- FBS, heat inactivated
- Gentamicin sulfate (50 mg/mL)
- Amphotericin B (Fungizone) (250 ug/mL)
- Sheep blood agar plate (or equivalent) for quality control
Worm media
Worm media kdorfman Fri, 04/01/2016 - 16:28Freezing medium
Freezing medium kdorfman Fri, 04/01/2016 - 16:30Per 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 - 18:11Per 1 L:
- 100 mL 10X M salts
- 300 µL 1M MgSO4
- water to 1 L
Filter sterilize
NGM
NGM kdorfman Fri, 04/01/2016 - 16:31Per 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.
put bottles on stir plate near sterile hood until handle-able
-
3.3 mL K2HPO4 + 21.7 mL KH2PO4 ↩︎
Yeast media
Yeast media kdorfman Mon, 02/08/2016 - 15:03284 Yeast Plate Code
284 Yeast Plate Code kdorfman Wed, 08/23/2023 - 15:49MV + Ade
MV + Ade kdorfman Wed, 10/18/2017 - 16:42For 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.
Mark with two blue lines
*Yeast Nitrogen Base without amino acids and ammonium sulfate
MV
MV kdorfman Mon, 02/08/2016 - 15:04Minimal Vitamin Medium for Bio 284
Autoclave an appropriately sized graduated cylinder to measure the glucose solution.
*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 - 16:39Plates 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 - 22:01Concentrate 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
Laney's SC high ade
Laney's SC high ade kdorfman Sun, 01/26/2020 - 16:22From 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 - 16:26SC -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 - 16:23Using 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.
Mark with one green line
SC-L high Ade from concentrate
SC-L high Ade from concentrate kdorfman Fri, 01/22/2021 - 20:55Mark with one green line.
SC-L high Ade mostly powder
SC-L high Ade mostly powder kdorfman Tue, 02/04/2020 - 13:41High 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 - 22:07SC-Leu low-Ade plates for Jeff Laney's cell and molecular biology lab
Laney's original
Laney's original kdorfman Sun, 01/26/2020 - 17:02SC-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 - 17:02SC -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 - 17:01Using 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.)
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 - 13:26High 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 - 21:45SC minus (adenine, histidine, leucine, tryptophan)
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 - 19:18Concentrate 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
SC-complete-high-Ade
SC-complete-high-Ade kdorfman Wed, 01/15/2020 - 22:12Liquid Medium
Using 4X SC-AHLT (because 10x won't go into solution):
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 - 13:33Recipe 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:
Mark with one green line
YEAD
YEAD kdorfman Fri, 08/04/2023 - 22:03Yeast 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.
YED
YED kdorfman Mon, 02/08/2016 - 19:22Yeast Extract Dextrose
For Bio 284
Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.
NOTE: 900 mL may boil over in a 1 L bottle in the autoclave. Try:
- Autoclave:
- YE + agar in 800 mL
- 100 mL H2O
- mix in BSC
- Add 100 mL sterile 20% glucose
- mix and pour
Mark with a single red line.
YEKAC
YEKAC kdorfman Mon, 02/08/2016 - 19:29Sporulation Medium for Bio 284
If you have concentrated potassium acetate solution, then:
- 20.4 mL 5M KOAc per liter
- 12.75 mL 8M KOAc per liter Fisher AAJ63372AE
Mark with a single black line.
If you are using potassium acetate powder, then:
YEPAD
YEPAD kdorfman Mon, 08/07/2023 - 17:05YEPAD 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
YEPAD for freezing
YEPAD for freezing kdorfman Wed, 08/16/2023 - 18:06Yeast 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.-
Grow a starter culture at 30 with shaking (250 rpm) until it reaches saturation.
In a 1.8 ml cryotube, mix 0.5 ml of the saturated culture with 0.5 ml of YPD containing 20% glycerol.
Flash freeze the tube in liquid nitrogen and store at -80.
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.
Allow the yeast to grow at 30 until colonies appear (2-6 days).
YNB + AS
YNB + AS kdorfman Wed, 01/15/2020 - 21:4410X 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 - 15:04Yeast extract-Peptone-Dextrose medium:
Dilutions
Dilutions kdorfman Mon, 03/07/2016 - 21:07Enter initial and final concentration (in matching units!), and final volume
Fixatives
Fixatives kdorfman Tue, 06/16/2009 - 17:53How to fix cells and tissues
Fixing adherent cells
Fixing adherent cells kdorfman Thu, 05/21/2020 - 17:39To 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 - 20:55Make in fume hood
3.7% formaldehyde
0.5% Triton X
in PBS
Formaldehyde stock: 37%
Triton X stock: 10%
Methanol fix
Methanol fix kdorfman Tue, 07/24/2012 - 17:05100% 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 - 16:565% paraformaldehyde
in PBS
Paraformaldehyde stock: 32%
Paraformaldehyde Fix
Paraformaldehyde Fix kdorfman Sun, 10/27/2013 - 19:323.7% paraformaldehyde, 0.5% Triton-X
in PBS
To fix the adherent cells (still in hood):
Paraformaldehyde stock: 32%
Triton X stock: 10%
EMS RT 15714
Paraformaldehyde from powder
Paraformaldehyde from powder kdorfman Wed, 03/03/2021 - 20:12Stir, heat to ~60C
Add NaOH until it clears, then bring to final volume
Paraglut
Paraglut kdorfman Mon, 09/24/2012 - 16:123.2% paraformaldehyde
0.1% glutaraldehyde
0.5% Triton X-100
in PBS
Bring to final volume with distilled water.
Gels
Gels kdorfman Thu, 05/24/2012 - 17:17EtBr gel
EtBr gel kdorfman Tue, 09/20/2022 - 15:02Ethidium bromide gel
*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
Gel Rigs
Gel Rigs kdorfman Wed, 01/11/2023 - 20:25brand | room | L x W (cm) | gel vol | # small wells | small well uL | # large wells | large well uL |
---|---|---|---|---|---|---|---|
IBI | 264 | 10 x 7 cm | 50 mL | 15 | 25 | 8 | 50 |
BioRad | 360, 364, 368 | 10 x 7 cm | 50 mL | 15 | 25 | 8 | 50 |
Labnet | genetics | 10 x 15 cm | 100 mL | 20 | 30 | 16 | 50 |
Sybr Safe gel
Sybr Safe gel kdorfman Tue, 09/20/2022 - 15:011 uL /10 mL gel
*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 - 21:25By compound
By compound kdorfman Mon, 04/23/2018 - 17:49ATP
ATP kdorfman Mon, 04/23/2018 - 17:5010 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 - 17:10Sigma 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
BMS
BMS kdorfman Fri, 03/25/2022 - 13:50BMS 833923
___ | ___ |
---|---|
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
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).
Sandhiya, S., Melvin, G., Kumar, S.S., et al. The dawn of hedgehog inhibitors: Vismodegib. J. Pharmacol. Pharmacother.4(1), 4-7 (2013).
Lin, T.L. and Matsui, W. Hedgehog pathway as a drug target: Smoothened inhibitors in development. OncoTargets and Therapy5, 47-58 (2012)
BP-A
BP-A kdorfman Mon, 03/22/2021 - 18:11Bisphenol A
Sigma 239658
MW 228.29
100 mM stock in DMSO = 0.023g/mL
BP-S
BP-S kdorfman Mon, 03/22/2021 - 18:12Sulfonyldiphenol
Sigma 103039
MW = 250.27
100 mM stock in DMSO = 0.025g/mL
Blebbistatin
Blebbistatin kdorfman Mon, 04/23/2018 - 17:49Myosin 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
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 - 20:48Fisher 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.
Caffeine
Caffeine kdorfman Tue, 04/13/2021 - 15:25MW 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 - 16:11Tetrahydrocannabinol solution 1 mg/mL in methanol
Sigma T464
store at 4C
Vial is in a white box in fridge door, room 362A
Centrinone
Centrinone kdorfman Thu, 06/01/2023 - 18:38PLK4 (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)
Con A coverslips
Con A coverslips kdorfman Sun, 05/24/2020 - 15:38Requirements: * 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 - 15:37Concanavalin-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 - 19:55CKD1 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.
Cycloheximide
Cycloheximide bcrcstaff Thu, 03/21/2019 - 13:28Protein 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 - 13:4310 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 - 18:21MW = 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
DAPT
DAPT kdorfman Fri, 03/25/2022 - 18:16Biological 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.
Estradiol
Estradiol kdorfman Thu, 03/24/2022 - 17:40β17-Estradiol
(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 RegistryNo | 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 - 17:15Sigma F1141
Attachment factor
GSA 10
GSA 10 kdorfman Thu, 01/28/2021 - 18:39Smo (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 - 18:08H 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
HU (Hydroxyurea)
HU (Hydroxyurea) kdorfman Wed, 01/06/2021 - 18:29ingredient | 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 - 20:06TCI 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.
Hydrocorisone
Hydrocorisone kdorfman Mon, 03/22/2021 - 18:06Fisher A16292 (Alfa Aesar) 1 g
1 mg/mL in 100% ethanol.
IGF-1R I
IGF-1R I kdorfman Fri, 03/25/2022 - 14:37Insulin-like growth factor-1 receptor inhibitor
Molecular Formula: C25H26ClN5O3
Formula Weight: 479.96
Solublity: 96 mg/mL in DMSO
IWP-2
IWP-2 kdorfman Fri, 03/25/2022 - 16:47Selleck 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 | - | - | - |
Importazole
Importazole kdorfman Thu, 05/10/2018 - 14:25401105-10mg 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 - 18:47Raises 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 - 16:29Low-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 - 18:16Laminin from mouse BD354232
1 mL 1 mM
Cell adhesion
Latrunculin
Latrunculin kdorfman Thu, 04/26/2018 - 17:46Alexis 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
ML-7
ML-7 kdorfman Wed, 04/25/2018 - 19:42ML-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
Melanocyte Stimulating Hormone
Melanocyte Stimulating Hormone kdorfman Wed, 04/25/2018 - 20:48Sigma 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]
Monastrol
Monastrol kdorfman Thu, 05/10/2018 - 15:17Inhibits kinesin 5
200 mM in DMSO
Final concentration 200 uM
5.5 uL aliquots
Monensin
Monensin kdorfman Wed, 04/25/2018 - 21:25Monensin 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
Nerve Growth Factor
Nerve Growth Factor kdorfman Fri, 05/11/2018 - 16:16Nerve 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 - 17:51Prevents 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 - 15:41Oxidopamine (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 |
PMSF
PMSF bcrcstaff Thu, 03/21/2019 - 14:14PhenylMethylSulfonyl Fluoride
Serine protease inhibitor
Sigma https://www.sigmaaldrich.com/catalog/product/sigma/p7626
Plant hormones
Plant hormones bcrcstaff Thu, 03/21/2019 - 14:49Compound | 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 - 17:04Non-specific attachment factor for cells; use it to coat coverslips.
Sigma P1524
- Add 50 ml of sterile tissue culture grade water to 5 mg of poly-lysine.
- Aseptically coat culture surface with 1 ml per 25 cm2 of solution. Rock gently to ensure even coating of the culture surface.
- After 5 minutes, remove solution by aspiration and thoroughly rinse surface with sterile tissue culture grade water.
- 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
Purmorphamine
Purmorphamine kdorfman Thu, 01/28/2021 - 18:25Tocris 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 - 17:09Fisher 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
Sodium alginate
Sodium alginate kdorfman Thu, 04/28/2022 - 14:52ALGINIC 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 - 17:40Taxol
Taxol kdorfman Mon, 04/23/2018 - 17:51Taxol (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)
Thyroid hormones
Thyroid hormones kdorfman Fri, 09/06/2019 - 18:38for 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
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)
Trichostatin A
Trichostatin A kdorfman Wed, 04/25/2018 - 19:52Cayman 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.
Valinomycin
Valinomycin kdorfman Wed, 04/25/2018 - 21:03Potassium 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.
Valproate
Valproate kdorfman Fri, 03/25/2022 - 15:34Sodium 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 - 17:53D3-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
By target
By target kdorfman Wed, 06/20/2018 - 21:28Target or Effect | Drug(s) |
---|---|
Vasoconstriction | Angiotensin |
Non-muscle myosin II ATPase | Blebbistatin |
Internal calcium levels | Bradykinin |
Nervous system | Cannabinol |
α-Mannose, α-Galactose sugars, glycoproteins and glycolipids | Concanavalin A |
cyclin-dependent kinase | cyclin inhibitor |
actin polymerization | cytochalasin D |
cell adhesion | fibronectin |
Golgi/ER | brefeldin |
Primers
Primers kdorfman Fri, 05/31/2013 - 17:35Primers 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 - 19:51Instant 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 - 20:47Acridine Orange
Acridine Orange kdorfman Fri, 07/27/2018 - 18:06Sigma 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 - 18:55Materials required but not in kit:
3% BSA in PBSTX
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
Buffer Additive
Buffer Additive kdorfman Thu, 03/26/2020 - 14:18To 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 - 17:0033mM (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 - 14:13Prepare 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 - 21:35DAPI 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 - 19:361 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 - 20:573-color LD + SYBR Safe
3-color LD + SYBR Safe kdorfman Mon, 10/15/2018 - 17:38The 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 - 21:01Ethidium 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 - 21:136X 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 - 21:04SYBR 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 - 21:13Safe-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 - 21:07Smart 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 - 16:44Invitrogen 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 - 15:34ER 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.
Fluo-4 AM
Fluo-4 AM kdorfman Thu, 04/26/2018 - 18:04Fluo-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.
plus HBS to final volume
Lysotracker
Lysotracker kdorfman Thu, 10/24/2019 - 20:17Lysotracker (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.
Mitotracker
Mitotracker kdorfman Wed, 02/21/2018 - 18:59Mitotracker GREEN
Mitotracker GREEN kdorfman Thu, 10/24/2019 - 20:34Excitation 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 - 20:34Invitrogen 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 - 14:32Invitrogen 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:
Minimal rinse! Very gently! Cold cells will fall off!
If following with ER tracker, rinse with HBSS, then replace with warm non-CO2 medium
If following with Mitotracker, rinse with PBS, then replace with warm non-CO2 medium
Nuc Blue
Nuc Blue kdorfman Thu, 07/14/2022 - 18:02Hoechst 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.
-
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 - 18:57CellLightTM Nucleus-RFP
Invitrogen (Thermo-Fisher) C10603
1 mL - received 10/26/23 - purchased for Bioimaging
Store in refrigerator
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
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 - 13:43Fluorescent 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 |
Phalloidin
Phalloidin kdorfman Thu, 08/13/2015 - 21:03General 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
FITC phalloidin
FITC phalloidin kdorfman Fri, 06/01/2018 - 18:40Thermo 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 - 18:42Alexa Fluor 568 Phalloidin
Alexa Fluor 568 Phalloidin kdorfman Fri, 06/01/2018 - 18:41Alexa 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 - 18:35Thermo 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 - 18:30Thermo 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 - 16:10Stains 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
Ph-HCL stain
Syto RNASelect
Syto RNASelect kdorfman Wed, 04/25/2018 - 19:47SYTO 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
TMRE
TMRE kdorfman Wed, 04/25/2018 - 21:17Tetramethrylrhodamine 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 - 19:06Thermo 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
Toluidine blue
Toluidine blue kdorfman Wed, 03/17/2021 - 16:12Toluidine 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.
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahU…
Transferrin
Transferrin kdorfman Thu, 05/10/2018 - 14:49Alexa-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.
Xylene Cyanol
Xylene Cyanol margaret Fri, 10/14/2011 - 15:59Stock solutions
Stock solutions kdorfman Fri, 10/28/2011 - 15:35ABA
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 - 17:0210 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 - 22:41Fisher 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 - 21:3910X 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 - 21:01Amphotericin
Amphotericin kdorfman Tue, 01/09/2018 - 18:29Anti-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 - 21:0250 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
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.
Chloramphenicol
Chloramphenicol kdorfman Thu, 04/23/2015 - 13:37Cipro
Cipro kdorfman Wed, 12/28/2011 - 21:02Ciprofloxacin 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
freeze
Doxycycline
Doxycycline kdorfman Mon, 01/18/2016 - 13:27Tetracycline category
solubility 50 mg/mL in water
disk has 30 ug
Erythromycin
Erythromycin kdorfman Fri, 01/20/2012 - 18:10solubility
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
-
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 - 21:0150 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
freeze
Kan calcs
Kan calcs kdorfman Tue, 10/02/2012 - 16:23Streptomycin
Streptomycin kdorfman Tue, 10/17/2023 - 19:43In 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 - 20:28Standard 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
-
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 - 21:02Irgasan
Sigma 72779-5g-f
MW 289.54
Boric acid
Boric acid kdorfman Tue, 12/20/2011 - 15:09Boric acid (H3BO3) 0.01 M (=10 mM)
1000x for M&S micronutrients
MW = 61.83
0.031 g/50 mL
Calcium chloride
Calcium chloride kdorfman Tue, 12/20/2011 - 15:01CaCl stocks in two concentrations:
MW (dihydrate) = 147
2.205 g/15 mL
7.35 g/50 mL
**Autoclave (or otherwise sterilize) for *C. elegans* medium**
1 Molar
1.1 g/15 mL
3.675 g/50 mL
**Autoclave (or otherwise sterilize) for *C. elegans* medium**
0.5 Molar (=500 mM)
Calcium nitrate
Calcium nitrate kdorfman Mon, 08/03/2015 - 18:47Ca(NO3)2.4H2O
FW = 236.1
0.4M stock:
94.4g/L = 0.189g/50mL
Chelated Iron
Chelated Iron kdorfman Fri, 01/12/2024 - 21:28FeSO4.7H2O 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 - 17:475 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 - 15:11Cobalt Chloride (CoCl2) 105 µM (=~ 0.1 mM)
10,000X for M&S micronutrients
MW (hexahydrate) = 237.93
0.00125 g/50 mL
DNA standards
DNA standards kdorfman Wed, 12/22/2021 - 21:38NEB MARKERS
NEB DNA standards (for general reference)
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)
DTT
DTT kdorfman Fri, 05/31/2013 - 17:21Dithiothreitol
Acros organics426380500 (store refrigerated in 362A) $98 at Fisher
154.25 MW
1M = 1.5425g in 10 mL H20
EDTA
EDTA kdorfman Tue, 12/20/2011 - 14:45EDTA (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
EGTA
EGTA kdorfman Fri, 08/09/2013 - 20:27EGTA 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
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 - 15:14Ferric 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
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 - 15:16Ferrous 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
Keep refrigerated
Turns yellow. Probably oxidation. Yellow comes off on a micropore filter.
Fluorescein
Fluorescein kdorfman Mon, 10/29/2018 - 17:44MW = 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
Histidine HCl
Histidine HCl kdorfman Wed, 01/15/2020 - 21:41100X 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 - 20:471 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 - 21:4310X 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 - 21:51LiOAc-SDS for DNA extraction from yeast
To make 100 mL of
Mix 20 mL LiOAc
with 5 mL SDS
and bring to final volume
and bring to final volume
Lithium Acetate
Lithium Acetate kdorfman Wed, 01/15/2020 - 21:26Lithium acetate dihydrate
C2H7LiO4
Fisher AA1341730
MW = 102.014
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 - 14:28LiCl 6M
MW = 42.39
Gets hot!!
MES
MES kdorfman Sun, 09/11/2016 - 17:47MW = 195.2
150 mM stock solution for LPGM
Magnesium chloride
Magnesium chloride kdorfman Tue, 12/20/2011 - 15:05Magnesium chloride (MgCl2) 1M
MW (hexahydrate) = 203.31
10.166 g/50 mL
20.33 g/100 mL
For Marine Biology (424) anesthetic reagent
for Aiptasia, make 0.37M in 30 - 35 PSU seawater
for Nematostella, dilute again 1:1
Magnesium sulfate
Magnesium sulfate kdorfman Tue, 12/20/2011 - 15:06Magnesium sulfate (MgSO4) 1M
MW (heptahydrate) = 246.48
Manganese chloride
Manganese chloride kdorfman Thu, 01/19/2012 - 07:30MnCl2 4H2O
M.W.197.9
5 mM
0.0495g/50mL
Manganese sulfate
Manganese sulfate kdorfman Tue, 12/20/2011 - 18:03MnSO4
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
Mannitol
Mannitol kdorfman Tue, 08/12/2014 - 17:45MW = 182.172
Potassium acetate
Potassium acetate kdorfman Tue, 12/20/2011 - 15:15Potassium acetate (C2H3O2K)
Fisher BP364-500 $46.30
MW = 98.14
49 g in 100 mL H2O
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 - 15:04Potassium chloride (KCl)
MW = 74.56
Potassium ferricyanide
Potassium ferricyanide kdorfman Tue, 12/20/2011 - 15:13Potassium ferricyanide (K3Fe(CN)6) 50 mM (= 0.05 M)
red salt
MW = 329.26
0.8232 g/50 mL
Store frozen
Potassium ferrocyanide
Potassium ferrocyanide kdorfman Tue, 12/20/2011 - 15:12Potassium ferrocyanide (K4Fe(CN)6) 50 mM (= 0.05M)
yellow salt
MW (trihydride) = 422.41
Store frozen
Potassium hydroxide
Potassium hydroxide kdorfman Thu, 01/19/2012 - 16:33KOH
MW = 56.11
Use for adjusting PIPES pH
Potassium nitrate
Potassium nitrate kdorfman Tue, 12/20/2011 - 15:18Potassium nitrate (KNO3)
Store in refrigerator
Potassium phosphate dibasic
Potassium phosphate dibasic kdorfman Sat, 01/14/2012 - 05:06K2HPO4
MW = 174.18
Potassium phosphate monobasic
Potassium phosphate monobasic kdorfman Tue, 12/20/2011 - 14:58Potassium phosphate monobasic (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 - 14:40SDS 20%
Sodium dodecanesulfate (=Sodium lauryl sulfate, NOT laureth)
CH3(CH2)11OSO3Na
DO NOT AUTOCLAVE - it (like all detergents) can boil over
Sodium acetate
Sodium acetate kdorfman Tue, 12/20/2011 - 15:08Sodium acetate (NaOAc) 3M
CH3COONa
MW (trihydrate) = 136.08
pH 5.2
Sodium azide
Sodium azide kdorfman Tue, 12/27/2011 - 18:08For PBS-Tw-Azide
NaN3
MW = 65
10% w/vol
Sodium bicarbonate
Sodium bicarbonate kdorfman Tue, 12/20/2011 - 14:54Sodium bicarbonate (CHNaO3) 0.5 M (=500 mM)
MW = 84.01
Write the date on it. Probably only stable for 2 weeks.
Sodium chloride
Sodium chloride kdorfman Tue, 12/20/2011 - 14:36Sodium Chloride (NaCl) 5 M
MW = 58.44
Sodium ferric EDTA
Sodium ferric EDTA kdorfman Mon, 08/03/2015 - 18:43NaFe(III)EDTA
FW = 367.05
50 mM
Sodium hydroxide
Sodium hydroxide kdorfman Mon, 10/29/2018 - 17:55NaOH
MW = ~40
10mM = 0.01M = 0.4g/L
Sodium phosphate dibasic
Sodium phosphate dibasic kdorfman Tue, 12/20/2011 - 15:00Use 0.2M for colorimetric exercise
Aliquote ~400 uL for a pair of students to each do the exercise
Use hot water
Sodium phosphate monobasic
Sodium phosphate monobasic kdorfman Tue, 12/20/2011 - 14:52Sodium 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.
Sodium sulfate
Sodium sulfate kdorfman Thu, 03/01/2012 - 14:17Na2SO4.10H2O
MW = 322.2
Spermidine
Spermidine kdorfman Fri, 05/31/2013 - 18:21Spermidine:
- 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 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
1 M stock
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.
20 mM student solutions
TBSTw
TBSTw kdorfman Thu, 11/17/2022 - 17:24Tris Buffered Saline with Tween
- Tris 20 mM
- NaCl 100 mM
- Tween 0.1%
- pH 7.4
Tris
Tris kdorfman Tue, 12/20/2011 - 14:42Tris 1M
Tris base MW = 121.14
pH to 7.5 or 8.0, depending on the application
Tris-HCl
Tris-HCl kdorfman Tue, 06/21/2022 - 17:26MW 157.6
Tris-HCl 1M
Triton X
Triton X kdorfman Tue, 12/20/2011 - 15:17Triton X 10%
MW = 646.86
1 g = ~1 mL
Tryptophan
Tryptophan kdorfman Wed, 01/15/2020 - 21:4250X 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 - 18:01Tween 10%
for PBS-Tween-Azide
1 g = ~1 mL [density = 1.095 g/mL at 25 °C (from Sigma website)]
Do not autoclave
Sugars
Sugars kdorfman Mon, 09/12/2016 - 21:45Glucose
Glucose kdorfman Mon, 09/12/2016 - 22:03Molarity (MW = 198.17)
Then bring to final volume. Filter sterilize
Percent
Then bring to final volume. Filter sterilize
Lactose
Lactose kdorfman Mon, 09/12/2016 - 22:07MW = 342.3
heat water first, then tap in lactose while stirring.
Mannitol
Mannitol kdorfman Mon, 09/12/2016 - 22:08MW = 182.17
Sucrose
Sucrose kdorfman Mon, 09/12/2016 - 22:02MW = 342.3
Solubility: 100 mg/mL in water at RT (= 10 g/100 mL)
Repairs
Repairs kdorfman Mon, 07/08/2013 - 18:15Emergency 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
MVI Microscope Service service@mvi-inc.com; 508-443-5234
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
BELIMED INCORPORATED
2325 CHARLESTON REGIONAL PKWY
CHARLESTON, South Carolina 29492 United States
Restriction Enzymes
Restriction Enzymes kdorfman Fri, 08/26/2022 - 15:50Updated Jan 2023
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" |
Routine checks 2022
Routine checks 2022 kdorfman Mon, 02/07/2022 - 18:42Safety Inspections
Safety Inspections kdorfman Tue, 05/29/2012 - 20:09hazardous waste pickup (sign into CEMS)
Suppliers
Suppliers margaret Thu, 10/13/2011 - 16:27Acros Organics
Acros Organics margaret Tue, 11/15/2011 - 15:25Addgene Inc.
Addgene Inc. margaret Thu, 10/13/2011 - 17:24Addgene
1 Kendall Square
Ste B7102
Cambridge, Ma
02139-1666
Phone:617-225-9000
Email: help@addgene.org
http://www.addgene.org/
Airgas
Airgas margaret Thu, 10/13/2011 - 17:18Airgas East
1361 Union Street
W. Springfield, Ma.
Phone:800-649-1639
413-781-6550
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 - 16:48Carolina Biological Supply Company
2700 York Rd.
Burlington, NC
27215-3398
Phone: 800-334-5551
Fax: 336-584-3399
Eppendorf
Eppendorf margaret Tue, 11/15/2011 - 15:27Fisher Scientific
Fisher Scientific margaret Tue, 11/15/2011 - 15:23Fisher custom oligos
Fisher custom oligos kdorfman Fri, 04/12/2013 - 16:57Fisher Scientific-Clonetech
Fisher Scientific-Clonetech margaret Thu, 10/13/2011 - 16:53Fisher 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
Krackler Scientific, Inc.
Krackler Scientific, Inc. margaret Thu, 10/13/2011 - 17:09Krackler Scientific
PO Box 1849
Albany, NY
12201-1849
Qiagen
Qiagen margaret Fri, 10/14/2011 - 15:45http://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
RNase-Free DNase
RNase-Free DNase margaret Fri, 10/14/2011 - 15:50Rainin
Rainin margaret Thu, 10/13/2011 - 16:31Rainin Road, Woburn Ma. 01888-4026
Phone: 781-935-3050
Order Line: 800-472-4646
Part numbers
Part numbers margaret Thu, 10/13/2011 - 16:43L-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
Scilogex
Scilogex margaret Tue, 11/15/2011 - 15:29Sigma-Aldrich
Sigma-Aldrich margaret Thu, 10/13/2011 - 17:13Sgma-Aldrich
3050 Spruce Street
St. Louis, MO
63103
Phone:Customer Service 800-325-3010
Technical Service 800-325-5832
Fax:800-325-5052
Thermo Fisher Scientific-Acros Organics
Thermo Fisher Scientific-Acros Organics margaret Fri, 10/14/2011 - 15:40Thermo 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
Thorlabs
Thorlabs margaret Tue, 11/15/2011 - 15:35Wahoo access
Wahoo access kdorfman Mon, 02/26/2018 - 18:34From your Mac
From your Mac kdorfman Wed, 02/27/2019 - 19:12On 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)