Difference between revisions of "20.109(F13): TA notes for module 2"
From Course Wiki
(→M2D6) |
MAXINE JONAS (Talk | contribs) m (1 revision: Transfer 20.109(F13) to HostGator) |
(No difference)
|
Latest revision as of 17:33, 29 July 2015
Contents
Day by Day Plan
M2D1
Before Class:
- Gather 96-well plates for pipetting exercise
- 2 plates / team = 9x2 + 8x2 = 34 plates (find 40 to be safe).
- 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:
- Help students with simulation exercise
- Stagger teams through pipetting exercise
M2D2
Before Class:
- 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.
- 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
- SKOV3 Media:
- Thaw and aliqout 1XTrypsin (2 mL x 20 for M2D3, store aliqouts at -20C)
RNA Harvest & RT Reactions
- Aliqout 1.1 mL of 0.5 mM EDTA for each group (20 total)
- Prepare Qiashredder & RNEasy columns
- Need 2 of each per team
- Total of 18 of each T/R
- Total of 16 of each W/F
- Need 2 of each per team
- 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)
- 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
- Resuspend primers from IDT in PCR grade water at 100 μM
- Aliquot into 10 uL aliqouts, one per team (20 total).
Day of Class
- Prepare RT MasterMix (2013: we will find the components during the W/F M2D1 class)
- Prepare & aliqout PCR MasterMix (2013: done -- 52 uL per tube)
- Pour 2% gels for analysis
M2D4
Before Class:
- Double check that there are 9 total SDS-PAGE gels.
- Make-up enough transfer buffer for each class.
- Make-up 1X TGS buffer -- will need ~ 1L for each box (5 boxes T/R and 4 boxes W/F)
- Make sure there are 20 aliqouts of protease inhibitor and phosphatase inhibitor
- Cut filter paper for WB -- Shannon will bring down from DAL lab
- Put ethanol bottles from transformations on the front bench.
- Have extra bag of eppendorf tubes ready on the front bench.
Day of Lab
- Serum starve 6-well plates by rinsing 1x with PBS and adding serum free media by 10am.
- Set-up gel boxes prior to start of lab (5 for T/R, 4 for W/F).
- Put PBS and lysis buffer on ice.
- Take the protein assay reagent out of the fridge and warm to RT.
- Get a Styrofoam cooler with dry ice to put leftover cell lysates.
- 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:
- 25 mL of ice-cold PBS
- one, 1 mL aliqout of lysis buffer (cold)
- very full ice bucket (need to use 4th floor ice machine + 3rd floor ice machine)
- plastic cell scraper
M2D6
Day before class:
For 2nd part Western blot analysis:
- 1L 10X TBS -- put on front bench w/ a 100 mL graduated cylinder.
- Put 3, 1L graduated cylinders on the front bench.
- Get Tween-20 from Shannon's bench in DAL lab -- put on front bench.
- Make-up more 50/50 OBB/PBS and store at 4C.
- Gather 9, 500 mL (or larger) glass bottles -- put on front bench.
For viability assay:
- 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).
- Prepare 45 mL M+D: M + 0.001% sterile DMSO (mix well).
- Aliqout 2.5 mL of M+D per team (labeled M+D).
- Prepare PC: M + 1% sterile DMSO (mix well)
- Aliqout 750 μL per team (labeled PC).
- Fill and autoclave all the 200 uL pipette boxes.
- Need ~80 eppendorf tubes --autoclave as needed.
- Sterilize with ethanol and put the multichannel pipettes in the TC room.
- Make sure we have enough troughs -- get from Shannon.
Evening before class:
For 2nd part Western blot analysis:
- Put membranes back into appropriate compartments
- 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
- Akt -- 1:2000
- pSTAT3 -- 1:1000
- STAT3 -- 1:2000
- The GAPDH antibody solution can be saved and used for the W/F section.
- Incubate overnight at 4C on Lauffenburger lab shaker
For viability analysis:
- Seed 10,000 cells/well in 100 μL per well in growth medium.
- Estimate by resuspending 1M cells in 10 mL per plate.
Day of class:
For 2nd part Western blot analysis:
- Put secondary antibodies on ice with cover on front bench
- Put OBB on front bench with hand held pipettor and 10 mL pipettes
- Make sure supplies for making TBS-T are on front bench
- You will take students to the Odyssey Licor in the Lauffenburger lab. Only two teams per trip to minimize distraction there.
For viability analysis:
- Shannon will make drug stocks after lecture.
- Each team will need 3 mL of Erlotinib (20 μM) -- Make up 27 mL total (27 mL M + 54 μL of 10 mM stock)
- Three teams will need 3 mL of LY294002 (40 μM) -- Make up 9 mL total (9 mL M + 7.2 μL of 50 mM stock)
- Three teams will need 3 mL of U0126 (20 μM) -- Make up 9 mL total (9 mL M + 9 μL of 20 mM stock)
- Three teams will need 3 mL of Sttatic (40 μM) -- Make up 9 mL total (7.2 μL of 50 mM stock)
- Two 96-well plates per team -- put on center bench.
- Put one trough in each hood.
- Warm up M, M+D, and PC aliqouts during pre-lab lecture
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
- BRET Buffer (M2D7): -- pre-make this now:
- PBS
- 0.1% Glucose
- 0.1% BSA
- 1mM Sodium Pyruvate
- Note: using sterile TC technique, aliquot what you need of the sodium pyruvate and then prepare the BRET Buffer in the main lab.
- Sterile filter using a bottle top filter and store at 4C.
- Transfer Buffer (10X) (without methanol) -- pre-make this now:
- 30.3 g Tris-Base
- 144 g Glycine
- Don't need to pH. Add dH2O to 1L.
- To make 1X: 100 mL 10X stock + 700 mL dH2O + 200 mL Methanol
- Transfer Buffer (1X) (M2D4): -- make this day of lab
- 25 mM Tris Base
- 192 mM Glycine
- 20%(v/v) Methanol
- CHO-K1 Growth Media:
- DMEM, high glucose
- 1% Sodium Pyruvate
- 1% NEAA
- 1% Glutamine
- 1% penicillin-streptomycin
- 10% FBS (Atlanta Biologicals)
- CHO-K1-EGFR-GFP Growth Media:
- CHO-K1 Media
- 50 μg/mL G418 (neomycin)
- CHO-K1 Starvation Media (can use for both K1 and EGFR-GFP cells):
- DMEM, high glucose
- 1%Sodium Pyruvate
- 1% NEAA
- 2% Glutamine
- 1% penicillin-streptomycin
- 0.1% FBS (Atlanta Biologicals)
- 0.35% BSA