20.109(F18): Prep notes for M1

M1D1

48 hr preceding laboratory:

• seed M059K (at 500K) and M059J (at 500K) in T25 flasks, 1 per team
• cell expansion schedule for D1, D3, and D4 prep

in tissue culture room (per team):

• 2 fresh T75 flasks
• 4.5 mL PBS
• 1.5 mL trypsin
• 25 mL media

in main lab (per team):

• not aliquoted: NMP stock, 1X PBS, gel bond film (pre-cut), markers, rulers

M1D2

• students will use cells seeded M1D1 for CometChip loading experiment

in tissue culture room (per team):

• 11 mL PBS
• 5 mL trypsin
• 11 mL media
• not aliquoted: trypan blue

in main lab (per team):

• not aliquoted: LMP solution

M1D3

48 hr preceding laboratory:

• seed M059K (at 1M) and M059J (at 1M) in T75 flasks, 1 per team
  • teaching faculty will harvest cells just prior to student use and concentrate to 500K / mL

in tissue culture room (per team):

• none

in main lab (per team):

• not aliquoted: 1X PBS, LMP solution, H₂O₂ (on ice at front bench), alkaline lysis buffer

M1D4

48 hr preceding laboratory:

• seed M059K (at 500K) and M059J (at 500K) in T75 flasks, 1 per team

in tissue culture room (per team):

• 11 mL PBS
• 5 mL trypsin
• 50 mL media

in main lab (per team):

• not aliquoted: unwinding / electrophoresis buffer, neutralization buffer

M1D5

in tissue culture (per team):

• 50 mL media
• 40 mL PBS
• not aliquoted: H₂O₂ (on ice at center bench)

in main lab (per team):

• none

M1D6

in tissue culture room (per team):

• none

in main lab (per team):

• 9 mL 1X TBS
• not aliquoted: Triton-X, BSA, anti-γH2AX antibody

students will prepare following solutions in class:

1. Triton-X solution = 2.5 mL 1X TBS containing 0.2% Triton-X
2. blocking solution = 2.5 mL 1X TBS containing 1% BSA
3. 1° antibody solution = 2.5 mL blocking solution containing 1:1000 dilution of anti-γH2AX antibody

M1D7

in tissue culture room (per team):

• none

in main lab (per team):

• none