20.109(F13): TA notes for module 2

Day by Day Plan

M2D1

Before Class:

1. Gather 96-well plates for pipetting exercise
   • 2 plates / team = 9x2 + 8x2 = 34 plates (find 40 to be safe).
2. Make Xylene Blue & Eosin Y solutions
   • Xylene Blue at 1mM
   • Eosin Y at 10 mM
   • 40 mL total of each will be enough for both days
   • Make 20 aliqouts of 1.8 mL each in 2 mL eppendorf tubes

During Class:

1. Help students with simulation exercise
2. Stagger teams through pipetting exercise

M2D2

Before Class:

1. Trypsinize and pellet one T150 flask of SKOV3 cells and 15 cm dish of HCC827
   • This should be done right before class start (or you may start right when the quiz is handed out).
   • Resuspend the pellet in PBS and aliquot into 9 and 8 eppendorfs for T/R and W/F, respectively, for each cell type.
   • Go ahead and use all the cells available to you, just report to the students how many cells are in the pellet.
   • Cells should be ok on ice in PBS until the are needed.

1. • SKOV3 Media:
     • McCoys 5A
     • 10% FBS (Atlanta Biologicals)
     • 1% Glutamine
     • 1% Pen-Strep
     • 1% Non-essential Amino Acids
     • Can prepare media ahead of time
   • Use 2 mL of total media per dish
2. Thaw and aliqout 1XTrypsin (2 mL x 20 for M2D3, store aliqouts at -20C)

RNA Harvest & RT Reactions

1. Aliqout 1.1 mL of 0.5 mM EDTA for each group (20 total)
2. Prepare Qiashredder & RNEasy columns
   • Need 2 of each per team
     • Total of 18 of each T/R
     • Total of 16 of each W/F
3. Prepare RLT + βME
   • Need 14.25 mL total volume (10 μL of BME / mL)
   • Prepare 375 μL aliqouts
   • Note: This could be done on Monday and stored at 4C until R/F -- do this in chemical hood (w/ safety glasses on)
4. Aliqout RNeasy Buffers (20 each)
   • 800 μL 70% Ethanol (see me for correct ethanol to use)
   • 1.45 mL RW1
   • 2.2 mL of RPE
   • 1 mL RNAse-free water

EGFR Mutation PCR

1. Resuspend primers from IDT in PCR grade water at 100 μM
2. Aliquot into 10 uL aliqouts, one per team (20 total).

Day of Class

1. Prepare RT MasterMix (2013: we will find the components during the W/F M2D1 class)
2. Prepare & aliqout PCR MasterMix (2013: done -- 52 uL per tube)
3. Pour 2% gels for analysis

M2D4

Before Class:

1. Double check that there are 9 total SDS-PAGE gels.
2. Make-up enough transfer buffer for each class.
3. Make-up 1X TGS buffer -- will need ~ 1L for each box (5 boxes T/R and 4 boxes W/F)
4. Make sure there are 20 aliqouts of protease inhibitor and phosphatase inhibitor
5. Cut filter paper for WB -- Shannon will bring down from DAL lab
6. Put ethanol bottles from transformations on the front bench.
7. Have extra bag of eppendorf tubes ready on the front bench.

Day of Lab

1. Serum starve 6-well plates by rinsing 1x with PBS and adding serum free media by 10am.
2. Set-up gel boxes prior to start of lab (5 for T/R, 4 for W/F).
3. Put PBS and lysis buffer on ice.
4. Take the protein assay reagent out of the fridge and warm to RT.
5. Get a Styrofoam cooler with dry ice to put leftover cell lysates.
6. Make-up Erlotinib solutions
   • Assuming 10 teams / section
   • Aliqout ~ 60 mL of serum-free media into a 100 mL media bottle
   • Add 50 uL of EGF solution (100 ug/mL) to the bottle and mix well
   • Add 10.9 mL of this solution to five, 15 mL conical tubes (1 uM, 0.1 uM, 0.01 uM, 0.001 uM, and 0 uM)
   • Add 12 mL to a 15 mL conical tube (10 uM)
   • To the 15 mL conical containing 12 mL, add 12 uL of 10 mM Erlotinib, mix well
   • Remove 110 uL from this conical tube and add to the 1 uM conical tube, mix well
   • serially dilute 10-fold down to 0.001 uM.
   • DO NOT add erlotinib to the final conical -- but instead, add 1.1 uL of DMSO and mix well)
   • Aliqout into 10, 1.1 mL aliqouts for the students

Each team will need:

1. 25 mL of ice-cold PBS
2. one, 1 mL aliqout of lysis buffer (cold)
3. very full ice bucket (need to use 4th floor ice machine + 3rd floor ice machine)
4. plastic cell scraper

M2D6

Day before class:

For 2nd part Western blot analysis:

1. 1L 10X TBS -- put on front bench w/ a 100 mL graduated cylinder.
2. Put 3, 1L graduated cylinders on the front bench.
3. Get Tween-20 from Shannon's bench in DAL lab -- put on front bench.
4. Make-up more 50/50 OBB/PBS and store at 4C.
5. Gather 9, 500 mL (or larger) glass bottles -- put on front bench.

For viability assay:

1. Prepare M: McCoys 5A + NEAA + Sodium pyruvate + P/S/G + 1% Serum (500 mL prepared Fall 2013)
   • Add 12.5 ng/mL EGF (for 500 mL = 62.5 μL of 100 ng/mL stock) -- mix well
   • Aliqout 14 mL of M per team (labeled M).
2. Prepare 45 mL M+D: M + 0.001% sterile DMSO (mix well).
   • Aliqout 2.5 mL of M+D per team (labeled M+D).
3. Prepare PC: M + 1% sterile DMSO (mix well)
   • Aliqout 750 μL per team (labeled PC).
4. Fill and autoclave all the 200 uL pipette boxes.
5. Need ~80 eppendorf tubes --autoclave as needed.
6. Sterilize with ethanol and put the multichannel pipettes in the TC room.
7. Make sure we have enough troughs -- get from Shannon.

Evening before class:

For 2nd part Western blot analysis:

1. Put membranes back into appropriate compartments
2. Use 5 mL of each antibody solution:
   • GAPDH -- 1:5000
   • EGFR -- 1:2000
   • pY1068-EGFR -- 1:1500
   • pERK -- 1:2000
   • ERK -- 1:2000
   • pAkt -- 1:2000
3. Akt -- 1:2000
   • pSTAT3 -- 1:1000
   • STAT3 -- 1:2000
   • The GAPDH antibody solution can be saved and used for the W/F section.
4. Incubate overnight at 4C on Lauffenburger lab shaker

For viability analysis:

1. Seed 10,000 cells/well in 100 μL per well in growth medium.
2. Estimate by resuspending 1M cells in 10 mL per plate.

Before the module starts

It will be easiest to pre-make many of the buffers that will be used during the module.

Old Notes

• Note: most of this is now out of date due to change in module aim

1. BRET Buffer (M2D7): -- pre-make this now:
   1. PBS
   2. 0.1% Glucose
   3. 0.1% BSA
   4. 1mM Sodium Pyruvate
   5. Note: using sterile TC technique, aliquot what you need of the sodium pyruvate and then prepare the BRET Buffer in the main lab.
   6. Sterile filter using a bottle top filter and store at 4C.
2. Transfer Buffer (10X) (without methanol) -- pre-make this now:
   1. 30.3 g Tris-Base
   2. 144 g Glycine
   3. Don't need to pH. Add dH2O to 1L.
   4. To make 1X: 100 mL 10X stock + 700 mL dH2O + 200 mL Methanol
3. Transfer Buffer (1X) (M2D4): -- make this day of lab
   1. 25 mM Tris Base
   2. 192 mM Glycine
   3. 20%(v/v) Methanol
4. CHO-K1 Growth Media:
   1. DMEM, high glucose
   2. 1% Sodium Pyruvate
   3. 1% NEAA
   4. 1% Glutamine
   5. 1% penicillin-streptomycin
   6. 10% FBS (Atlanta Biologicals)
5. CHO-K1-EGFR-GFP Growth Media:
   1. CHO-K1 Media
   2. 50 μg/mL G418 (neomycin)
6. CHO-K1 Starvation Media (can use for both K1 and EGFR-GFP cells):
   1. DMEM, high glucose
   2. 1%Sodium Pyruvate
   3. 1% NEAA
   4. 2% Glutamine
   5. 1% penicillin-streptomycin
   6. 0.1% FBS (Atlanta Biologicals)
   7. 0.35% BSA