20.109(F19): Prep notes for M1

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):

• 135 μL 12 mM MMS
• 8 μL 100 mM As
• 45 mL PBS
• 40 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:

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

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

• teaching faculty will prepare CometChips, 1 per team

in tissue culture room (per team):

• none

in main lab (per team):

• 50 μL 12 mM MMS
• 10 μL 100 mM As
• 12 mL media

• not aliquoted: 1X PBS, LMP solution, alkaline lysis buffer

M1D5

in tissue culture (per team):

• none

in main lab (per team):

• not aliquoted: unwinding / electrophoresis buffer, neutralization buffer, SYBR gold

M1D6

in tissue culture room (per team):

• none

in main lab (per team):

• none

M1D7

in tissue culture room (per team):

• none

in main lab (per team):

• none