20.109(F19): Prep notes for M1
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M1D1
48 hr preceding laboratory:
- seed CHO (350K) in T75 flasks, 1 per team
- cell expansion schedule for D1, D3, and D4 prep
in tissue culture room (per team):
- 1 fresh 12-well plate
- 5.5 mL PBS
- 2.5 mL trypsin
- 35 mL media
- not aliquoted: trypan blue
in main lab (per team):
- none
M1D2
in tissue culture room (per team):
- X μL 12 mM MMS
- X μL 100 mM As
- X mL PBS
- X mL media
- not aliquoted: EdU
in main lab (per team):
- not aliquoted: NMP stock, 1X PBS, gel bond film (pre-cut), markers, rulers
M1D3
48 hr preceding laboratory:
- seed CHO (350K) in T75 flasks, 1 per team
- teaching faculty will harvest cells just prior to student use and concentrate to 500K / mL
prior to laboratory
- teaching faculty will prepare Click-iT reaction mix
in tissue culture room (per team):
- none
in main lab (per team):
- 9 mL 1X TBS
- not aliquoted: Triton-X, BSA, anti-γH2AX antibody, mounting media, 1X PBS, LMP solution
students will prepare following solutions in class:
- Triton-X solution = 2.5 mL 1X TBS containing 0.2% Triton-X
- blocking solution = 2.5 mL 1X TBS containing 1% BSA
- 1° antibody solution = 2.5 mL blocking solution containing 1:1000 dilution of anti-γH2AX antibody
M1D4
48 hr preceding laboratory:
- seed CHO (350K) in T75 flasks, 1 per team
- teaching faculty will harvest cells just prior to student use and concentrate to 500K / mL
prior to laboratory
- prepare CometChips, 1 per team
in tissue culture room (per team):
- 11 mL PBS
- 5 mL trypsin
- 50 mL media
in main lab (per team):
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:
- Triton-X solution = 2.5 mL 1X TBS containing 0.2% Triton-X
- blocking solution = 2.5 mL 1X TBS containing 1% BSA
- 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