Model Systems

Model Systems kdorfman Tue, 07/03/2012 - 17:26

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Cells 2012

Cells 2012 kdorfman Tue, 07/03/2012 - 17:26

Lab 1: FL microscope

Lab 1: FL microscope kdorfman Mon, 07/30/2012 - 18:03

10 slides w/ DAPI

(1 per microscope + 2 extra)

Micrometers

Lab 2: Organelles

Lab 2: Organelles kdorfman Mon, 07/30/2012 - 18:26

5 or 6 slides each:

  • Rhodamine Phalloidin "1G"
  • Stained for rat anti-tubulin + goat anti-rat FITC (or GFP-tubulin cells) "2B"
  • Acridine orange stained for nucleoli "3G"
  • Stained with with mouse anti-Golgi + goat anti-mouse 568 "4G" (formaldehyde fixed)
  • Stained with rabbit anti-ZO1 + goat anti-rabbit Cy (cell-junction) "5G"

Lab 3: live cells; staining

Lab 3: live cells; staining kdorfman Mon, 07/30/2012 - 18:31

20 fixed coverslips for phalloidin staining

3 MatTeks per group for mitotracker and ?

aliquots of

  • phalloidin
  • mitotracker
  • non-CO2 medium

Lab 4 - discussion

Lab 4 - discussion kdorfman Mon, 07/30/2012 - 20:15

Finish looking at slides, discuss cell division

Lab 5: mitosis

Lab 5: mitosis kdorfman Mon, 07/30/2012 - 20:18

10 slides DAPI + tubulin

2 MatTeks per group each:

  • LLCPK with GFP tubulin
  • LLCPK parentals

NUC-Blue for staining chromosomes in live cells (Invitrogen R37605 or Fisher NC0291762), 1 bottle per station

  • Remove from culture dish after ~10 minutes. Replace stain medium with fresh medium. Otherwise it gets brighter and brighter as movie goes on

Non-CO2 medium

Lab 6: Mitosis

Lab 6: Mitosis kdorfman Mon, 07/30/2012 - 20:45

4 MatTeks per group of LL-GFP-tubulin

Double-strength inhibitors, made up in non-CO2 medium:

  • STLC Sigma 164739 (Fisher 50-703-1833), MW = 363.47

  • need 1 mL aliquots 10 µM in non-CO2 medium for students

  • Make 100 mM stock:

    • 1 g in bottle.
    • Mix with 27.5 mL DMSO
    • heat to 65C (~1 hr), vortex
    • if necessary, filter sterilize to remove insoluble particles.
    • save as 1 mL aliquots. (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 and vortex to redissolve it.
  • Make 17 mL 10 µM in medium for students

    • 170 µL 1 mM stock
    • medium to 17 mL
    • aliquot
  • Make 10 µL aliquots of 1 mM stock

    • label says to add 990 µL medium to make 10 µM working solution

from Alyssa:madke STLC in DMSO and that she heated to 42C, and vortexed. She says it went from clear to yellow/brown. we use the reagent at micromolar - 1-10 µM. Her stock solution was 1mM.

students will be adding 1 mL of inhibitor to 1 mL medium in dish; the ready-to-use 1 mL (in medium) should be at least 2µM

  • Taxol (20 µM final)
  • soluble in DMSO, not water.
    • students pick either Taxol or nocodazole, so make only 10 each
    • each tube has 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 in two tubes!
    • mix with medium in 15 mL conical to make 10 mL at 20 µM
  • Nocodazole (3.3 µM final)

    • each tube has 3 µL 33mM
    • add 97 µL to tube to make 100 µL of 1 mM
    • 33 µL 1mM + 10 mL medium = 3.3 µM
  • Nuc Blue from Invitrogen (or Fisher NC0291762), 1 bottle per station

    • Remove from culture dish after ~10 minutes. Replace stain medium with fresh medium. Otherwise it gets brighter and brighter as movie goes on

Lab 7: Cytokinesis

Lab 7: Cytokinesis kdorfman Mon, 07/30/2012 - 20:58

Myosin & tubulin expressing cells

2 MatTeks each per group

For ML-7, the final conc should be 75uM, so the 2X needs to be 150uM. For the Jasplak: the final conc should be 7uM so the 2X needs to be 14uM (but if 15 is easier, go with it). we use latrunculin at 5uM, and I think cytoD should be similar (I think I previously gave a range for cytoD of 1-10uM). Let's go with 5, so the 2X would be 10uM.

Inhibitors in non-CO2 medium (1 mL aliquots) each group does one (so make 5 of each):

  • cytochalasin D Sigma C2618 or C8273

    • tubes have 40 µL 2.5 mM
    • label says add 460 µL to make 500 µL of 200 µM
    • need 10 µM (2x final working concentration)
    • 20 µL 2.5 mM
    • 5 mL non0CO2 medium
    • gives 5 1 mL aliquots at 10 µM
  • Jasplakinolide – Santa Cruz biochemical, product sc202191 F

    • inducer of actin polymerization & stabilization
    • 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
  • ML-7 Sigma, 12764.

    • 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

Lab 8: projects

Lab 8: projects kdorfman Mon, 07/30/2012 - 21:04

Students limited to 3 MatTeks of one of the following:

  • GFP tubulin
  • tubulin-myosin cells

Plus their choice of inhibitors:

  • latrunculin
  • taxol
  • nocodazole
  • jasplakinolide
  • ML-7
  • STLC

Lab 9: Reports

Lab 9: Reports kdorfman Mon, 07/30/2012 - 21:08

Report on mitosis experiments

RNA

RNA kdorfman Tue, 07/03/2012 - 17:40

Acridine orange

Acridine orange kdorfman Fri, 08/03/2012 - 21:01

Sigma A8097-10 mL

10 mg/mL = 33mM

MW = 301.81

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.

SYTO RNASelec

SYTO RNASelec kdorfman Fri, 08/03/2012 - 20:59

SYTO RNASelect Green Fluorescent Cell Stain (Invitrogen S32703)

$210 from Invitrogen

Fluoresces green when bound to RNA.

excite: 490 nm, emit: 530 nm

Can be used in live or fixed cells. Fix in methanol, NOT formaldehyde!

Don't use in conjunction with red-orange dyes.

Stock:

100µL 5 mM in DMSO. Store at -20C, dessicated, dark.

To thaw: warm to RT, spin down.

Should be stable for >= 1 year.

Make labeling solution

Make 5µM intermediate stock:

2 µL 5mM stock + 1998 µL medium or PBS

Make 20 100 µL aliquots in 1.5 mL tubes

Label: RNASelect - 100 µL - 5 µM intermediate stock in PBS; Add 900 µL to make labeling solution

Make 500 nM labeling solution in medium or PBS

100 µL 5 µM intermediate + 900 µL medium

Protocol

Live cells

  • Cells on coverslip
  • Warm 500 nM labeling solution to 37C
  • Incubate at 37C 20 min
  • Rinse twice in PBS or medium
  • Add warm medium, let cells rest 5 min
  • Fix in chilled methanol 10 min at -20C
  • Several washes in PBS

Fixed cells

  • Remove coverslip from medium
  • Fix in chilled methanol 10 min at -20C
  • Remove methanol, let slip sit in PBS 5 min
  • Apply labeling solution 20 min RT
  • Wash 5 min in PBS
  • Mount coverslip

Cells 2018

Cells 2018 kdorfman Wed, 01/03/2018 - 17:07
Date Topic cells needed
1/31 microscopy boot camp 7 fixed, stained slides
3/30 introduction to cultured cell model; Demo of sterile technique, plating etc 3 flasks of LLCs to split & plate on MatTeks; 6 MatTeks live LL-GFP-alpha tubulin/red myosin cells to view
4/4 Learn to stain cells; 10 paraGlut fixed coverslips for staining
4/4 Nucleofect (pat has siRNA) 3 fresh flasks for nucleofection, plated Tuesday with 10^6 cells; 6 coverslips of live cells for fixing
4/6 fix the siRNA cells no cells; fixative
look at stained cells from 4/4
look at stained organelles use the stained slides bioimaging
4/11 mitosis. 12 Live GFP tubulin cells in dishes, fixed llcpk, microtubule stain, for mitotic index, fixed at 24 and 48 hr
4/13 mitosis: inhibitors 12 live GFP tub in dishes, 12 live parental on coverslips for treat, fix
4/18 quantify data from 4/13; live cell + inhibitor experiment Live cells in Matteks, plated Monday
4/20 presentation for module

Cells 2022

Cells 2022 kdorfman Mon, 02/14/2022 - 17:05
Date Class # Due before class In class activities Due at end of class
2/28 MON 1 -- Mammalian cells as a model system. Identify stages of mitosis Determine mitotic index Group contract Saved images of mitosis
3/2 WED 2 Forum post #1 (20pts) Learn cell culture Make Fig w/ marker bar. Live cell imaging!
3/7 MON 3 Figure of mitosis/MI (35pts) Immunofluorescence Set up growth curve Protocol: cell culture (30pts)
3/9 WED 4 Protocol: fixation and staining (30pts) Finish Immuno/image Experimental design Finish growth curve Experimental plan - DRAFT
3/14 SPRING BREAK ----- ------ ------
3/16 SPRING BREAK ----- ----- -------
3/21 MON 5 Forum post #2 (20pts) Group project: mitotic inhibitors Data set for cell growth (35pts)
3/23 WED 6 Group project: mitotic inhibitors Exptl Plan for next class (15 pts)
3/28 MON 7 Group project: mitotic inhibitors Draft presentation
3/30 WED 8 In Class presentations Class & grp eval (5pts) Presentation: (40pts) Indiv exptl summary due 4/4 (20pts)

Fish 2012

Fish 2012 kdorfman Thu, 09/13/2012 - 15:00

materials

materials kdorfman Thu, 09/13/2012 - 15:03
  • no. 5 forceps
    • Fisher S95311 Straight; Ultra fine pointed tips; Aven Tools 18056ER $12.53
    • Digikey 243-1099-ND Aven Tools 18062-ER $15.65
    • Digikey 243-1054-ND Aven Tools 18062EZ $5.75 (green)
    • 243-1051-ND 18056EZ (turquoise) sharpest!
    • 243-1053-ND 18013EZ (yellow)

Forceps tip sizes

  • methyl cellulose 3% ~200 mL > Flinn Scientific M0171 $7.55 100 mL

It takes ~ a week of stirring to make the powder ( Fisher S80080 100G) go into solution at this concentration!

  • Fish water (get a carboy full from Craig)

  • Lithium Chloride (MW = 42.4) solutions:

    • 10M LiCl in 10 mL H2O (4.24 g) (vortex in 15 mL conical. gets hot.) Check stock solutions first

    • 0.3 M LiCl in fish water (1.272 g/100 mL)

    1 µL 10 M/1 mL fish water

  • deep depression slides (Pyrex 9-spot plates)

  • 60 mm petri dishes - CellTreat with gripping ring are good for this (sterility not necessary)

  • glass-bottom 29 mm petri dishes (InVitro Scientific)

  • 2 packs (2 x 48) Netwell baskets (Corning 3477)

Fisher 07-200-211 $215.55

  • 12 well plates (for the baskets to fit in)

  • timers

  • superglue

  • short tip pasteur pipettes

  • fishing line

  • transfer pipets (large, non-sterile)

  • mirror base dissecting microscope

Fish 2014

Fish 2014 kdorfman Thu, 03/20/2014 - 19:03

Drugs for projects

Drug vendor part # amt price
SU5402 (FGF inhibitor) Sigma SML0443-5MG 5 mg $83.60
Dorsomorphin (BMP Inhibitor) [6-(4-(2-PIPERIDIN-1-YLETHOXY)PHENYL)-3-P] Sigma P5499-5MG 5 mg $151
IWR-1 (WNT inhibitor) Sigma I0161-5MG 5 mg $70.10
Rapamycin Fisher 502307198 100 ug $57
Isoliquiritigenin Fisher 506850 10 mg $206.55
DMSO
Ethidium bromide

Fish 2019

Fish 2019 bcrcstaff Tue, 03/05/2019 - 15:37
Dates: Lab exercise: Lecture: Assignment due:
Tues 2/26 Introduction; Make your own tools Principles of development
Thur 2/28 Developmental staging of wild-type & mutant fish; Reporter lines. Zebrafish embryology PBL #1 posted
Tues 3/4 What’s in your wallet? BPA or BPS? Which is worse? Environmental toxins & developmental biology PBL #1 due (deliver to Craig via email)
Thur 3/6 BPA & BPS wrap-up. 500ug/mL, 50 ug/mL BPA and BPS stock solutions PBL #2 posted
Tues 3/11 NO CLASS; SPRINGBREAK!
Thur 3/13 NO CLASS; SPRINGBREAK!
Tues 3/18 Bones & teeth - Imaging Zebrafish skeletal & dental anatomy PBL #2 due (deliver to Craig via email)
Thur 3/20 Bones & teeth - Measuring Morphometrics PBL #3 posted
DUE 3/21: Turn in experimental plan for independent research (deliver to Craig via email).
Tues 3/25 Independent Research! PBL #3 due (deliver to Dylan via email)
Thur 3/27 Independent Research!
Tues 4/2 Present your data to class Turn lab notebooks in to Dylan & send presentations to Craig via email.

Fish 2022

Fish 2022 kdorfman Tue, 03/22/2022 - 16:10

Craig Albertson, starting April 4

  • 3% methylcellulose - 1 or 2 of those ~250ml containers should be more than enough.

  • 80% glycerol (mixed in 1% PBS)

  • 2 boxes of wooden pencils (~50 total) - the brand doesn't matter, they just need to have an eraser.

  • Small sewing needles - Smallest size is like 80 (12) I think. If in doubt maybe get an assortment? they will be inserted into the pencil erasers - the small needles are generally finer than most of the probes we have in teaching labs.

  • Insect needles - These will also be inserted into erasers for more fine manipulation/dissection of fishes. The goal will be for each group to have one "fine" and one "course" probe.

Flies 2012

Flies 2012 kdorfman Fri, 09/14/2012 - 22:02

Wiremold CDG-50 Cord Protector 50 ft Amazon $84.59

cut pieces are stored above the white board in 360

CO2 tanks from AirGas: Place order for 3 tanks, with no returns.

Fly brushes: Princeton Art & Brush Co 4350R-0 round

http://www.davinciartistsupply.com
4350R Short Handle - Round 0 36 @ $1.76 = $63.36

NightSea blue flashlghts & Yellow filters

From Kegworks.com (quote from phone order)

In-Line Ball Valve Shut Off, 1/4” Barb: 24 @ 4.50, total 108.00
$12.07 shipping. total $120.07
1460 Military Road, Rear Building
Kenmore, NY 14217
P: 716.362.9212
F: 716.856.9684

Flies 2016

Flies 2016 kdorfman Wed, 02/24/2016 - 17:20

Naegleria 2018

Naegleria 2018 kdorfman Tue, 01/16/2018 - 21:59

I would think 6 plates per student should be sufficient for whatever protocol they devise. We are up to 12 students now, so maybe 3 sleeves of plates?

To make Medium 21, add the following in order to a liter flask,

DIW (pure deionized water) 800 ml

K2HPO4 (anhydrous) 0.72 g

KH2PO4 (anhydrous) 2.16 g

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.

Naegleria 2019

Naegleria 2019 bcrcstaff Fri, 01/04/2019 - 16:48

Naegleria 2019 schedule

Naegleria 2019 schedule bcrcstaff Fri, 01/04/2019 - 16:51
date activities supplies
1/22 discussion none
1/24 Microscopy boot camp slides for observation?
1/29 Cloning bootcamp AgeI
rSAP
Ligase kit
Qiagen PCR Purification
37C, 42 C heat block
liquid LB
LB amp (carb?) plates (3/pair), sterile glass beads
Competent cells (DH5 alpha, just for cloning)
1/31 Naegleria 101 HT115 (from Lil?), Lugol's
loops
2 mM tris pH 7.6
30C shaking incubator
sterile toothpicks
liquid LB, carb or amp stock,
coverslip bottom dishes
sterile 10 mL culture tubes
2/5 Cloning bootcamp 2
transform dsRNA-expressing bacteria + controls
miniprep
restriction enzymes (which ones?)
1.2 % agarose gel (wide)
TAE
gel rig & viewer
20 carb/tet LB plates
2/7 start ON dsRNA bacteria cultures, prep Naegleria cultures induction medium
LB Amp 2 tubes of 5 mL/group
2/11 plate ON cultures NM Amp/IPTG ( 0.5 - 1 mM) plates 2/ pair
beads
2/12 grow bacteria. Plate lawns on induction medium Naegleria & E. coli growth plates
sterile beads
2/14 differentiate fed amoebae and controls iodine (Lugols?)
2mM Tris
culture tubes
shaking incubator
coverglass bottom dishes
2/21 presentations none

https://wahoo.cns.umass.edu/node/723 NM

Naegleria 2022

Naegleria 2022 kdorfman Fri, 12/17/2021 - 21:18

planning page

schedule ((Digest+CIP, column purify, ligation, transformation). Flowcharts+review+revision**

date # topic
1/26 1 Intro
1/31 2 Microscope bootcamp
DAPI slides, fluorescence microscopy power point
2/2 3 2/2 | 3 | Cloning 1
((Digest+CIP, column purify, ligation, transformation). Flowcharts+review+revision
2/7 4 Naegleria 101
2/9 5 Cloning 2
2/14 6 Experiment 1
2/16 7 Experiment 2
2/22 8 Experiment 3
2/23 9 Wrap up

Competent HT115

Competent HT115 kdorfman Mon, 12/20/2021 - 15:59
to make: aliquots
at this volume: uL
Innoculate mL LB tet
with mL overnight culture
Grow to OD595=0.4
Spin 3000rpm for 10 min at 4C
Resuspend in: mL cold 50mM CaCl2 GENTLY
Spin as before, 3000rpm for 10 min at 4C
Resuspend in: mL cold 50mM CaCl2 GENTLY
Use immediately or freeze by adding: mL sterile glycerol

  • Inoculate overnight culture in LB + antibiotic (TET for HT115(DE3) strain) (2-5ml). Shake overnight at 37C.

  • Inoculate 25 ml LB + antibiotic with overnight culture, 1:100 dilution. Grow cells to OD595= 0.4. Can grow cells in 50 ml sterile centrifuge tube.

  • Spin cells 10 min 3000 rpm at 4C.

  • Resuspend pellet in 0.5X original volume cold, sterile 50 mM CaCl2 (12.5ml). Resuspend by GENTLY pipetting up and down a few times with a wide bore pipet--no vortexing.

  • Incubate on ice 30 min.

  • Spin as before at 4C.

  • Resuspend pellet as before in 0.1X original volume CaCl2 (2.5ml). Keep cells cold (4C).

  • Use 50-200 ul for transformation. Cells can be used as is for up to three days (stored at 4C).

  • The cells can be frozen by: adding glycerol to final concentration of 10%, rapid freezing on dry ice/EtOH, and storing at –80.

Naegleria 3

Naegleria 3 kdorfman Sat, 12/18/2021 - 12:00

Cloning Bootcamp

  • 37C heatblock
  • 42C heatblock
  • Age1
  • 10X buffer NEB1.1
  • rSAP
  • Sterile water for cloning
  • Ligase + 2X rapid lligase buffer
  • 1 sample: PCR cleanup kit (Qiaquick) (one per group)
  • 1 tube (5 rxns) Competent cells (DH5 alpha, just for cloning)
  • 5x LB+Amp plates
  • Sterile beads for spreading
  • 30 ng/ul backbone plasmid for cutting (estimated 15n/ul after elution w/ 30ul)
  • Gel purified inserts (gamma tub, centrin, sas-6 at 15ng/ul)
  • LB (plain) for transformation recovery

Naegleria 4

Naegleria 4 kdorfman Sat, 12/18/2021 - 12:37

Naegleria 101 (2/7/22)

Activities:

  • Differentiate cells,
  • watch amoebae (phase microscopy),
  • look at flagellates.
  • Pick colonies for cloning.

Materials:

  • 4 LB+ Amp +
  • Tubes for growing cultures from previous lab’s cloning
  • Toothpicks for above

Naegleria 5

Naegleria 5 kdorfman Tue, 02/08/2022 - 21:08

8 QIAprep Spin miniprep kits for 4 cultures each

Notes before starting

  • Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000.
  • Add the provided RNase A solution to Buffer P1, mix and store at 2–8°C.
  • Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
  • All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge.

Each kit contains:

  • 4 spin columns
  • 6 buffers, as below
Buffer uL per rxn mL per 4.5 rxns
P1 250 1.125
P2 250 1.125
N3 350 1.575
PB 500 2.25
PE 750 3.375
EB 50 0.225

Other materials

Equipment

  • Microcentrifuges capable of 13000rpm
  • Hot blocks at
    • 37
    • 42
  • two wide gel rigs
  • two power supplies
  • two blue light boxes with photography box

Naegleria 6

Naegleria 6 kdorfman Thu, 02/10/2022 - 14:06

M Feb 14

32 (?) tubes of LB-amp

Yeast 2014

Yeast 2014 kdorfman Fri, 01/10/2014 - 18:08

Wei-Lih Lee

Yeast 2015

Yeast 2015 kdorfman Fri, 01/16/2015 - 19:17

1/22 Th

  • slides,
  • coverslips
  • razor blades
  • scotch tape
  • valap
  • heat block (6)
  • microfuge (6 24 slot)
  • 50 mL conicals
  • 15 mL conicals
  • 200 mL beakers (1 per group)
  • Q-tips (long)

Tu 1/27/15

Tu 1/27/15 kdorfman Thu, 01/22/2015 - 16:40

*4 PCR machines with dual blocks - enough for eight groups of students

*8 DNA gel apparatus

*3 weighing balance for pipetting exercise

*10 mM dNTPs, 60 microliters total

*Phusion DNA polymerase, 10 microliters total (we prefer Phusion from NEB because we are amplifying with 80 nt long primers. I don’t recall if your freezer has any of this DNA polymerase. If not, I’m willing to just use the one in my lab)

For Thursday, Jan 29th:

MBOMS will run gel and need to coordinate with other labs to use the gel doc.