20.109(F13): TA notes for module 2

Before the module starts

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

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