20.109(S19): Prep notes for M2

M2D1

48 hr preceding laboratory:

• seed DLD-1 (at 250K) and BRCA2-/2 (at 500K) in T25 flasks, 1 per team
• cell expansion schedule for D1, D2, and D8 prep

in tissue culture room (per team):

• 2 fresh T25 flasks
• 5 mL PBS
• 3 mL trypsin
• 12 mL media

in main lab (per team):

• none

M2D2

24 hr preceding laboratory:

• seed DLD-1 (at 500K) and BRCA2-/- (at 1M) in T75 flasks, 2 per team

in tissue culture room (per team):

• 22 mL PBS
• 50 mL media
• 15 μL etoposide

in main lab (per team):

• none

M2D3

prior to laboratory:

• prepare aliquots of purified RNA to supplement student samples

in tissue culture room (per team):

• 35 mL PBS
• 9 mL trypsin

in main lab (per team):

• RNA purification aliquots
  • 1.5 mL RLT
  • 1.5 mL 70% EtOH
  • 3.0 mL RW1
  • 4.2 mL RPE
  • 140 μL RNase-free H₂O

• cDNA aliquots
  • 40 μL DEPC-treated H₂O
  • other reagents will be kept at front laboratory bench; students will prepare master mix with filtered tips

M2D4

in tissue culture room (per team):

• none

in main lab (per team):

• none

M2D5

prior to laboratory:

• prepare aliquots of cDNA to supplement student samples

in tissue culture room (per team):

• none

in main lab (per team):

• 2.75 mL RNase-free H₂O
• qPCR aliquots
  • 420 μL Buffer PB
  • 1.5 mL Buffer PE
• 30 μL GAPDH primer mix
• 30 μL p21 primer mix

M2D6

in tissue culture room (per team):

• none

in main lab (per team):

• none

M2D7

in tissue culture room (per team):

• none

in main lab (per team):

• none

M2D8

24 hr preceding laboratory:

• seed DLD-1 (6 wells at 25K) and BRCA2-/- (6 wells at 50K) in 12-well plate, 1 per team

in tissue culture room (per team):

• 28 mL PBS
• 18 mL media (for etoposide treatment)
• 20 μL etoposide
• 50 mL media (for drug treatment)
• 10 μL loperamide
• 10 μL mibefradil

in main lab (per team):

• none

M2D9

in tissue culture room (per team):

• none

in main lab (per team):

• 6.5 mL CellTitre Glo reagent
• 30 mL PBS
• 6.5 mL media