Difference between revisions of "20.109(S18): Prep notes for M2"
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Noreen Lyell (Talk | contribs) (→M2D3) |
Noreen Lyell (Talk | contribs) (→M2D1) |
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'''48 hr preceding laboratory:''' | '''48 hr preceding laboratory:''' | ||
*seed DLD-1 (at 250K) and BRCA2-/2 (at 500K) in T25 flasks, 1 per team | *seed DLD-1 (at 250K) and BRCA2-/2 (at 500K) in T25 flasks, 1 per team | ||
− | *[[Media:Sp18 M2 cell seeding schedule.xlsx|cell expansion schedule]] for D1, D2, and D8 prep | + | *[[Media:Sp18 M2 cell seeding schedule v2.xlsx|cell expansion schedule]] for D1, D2, and D8 prep |
'''in tissue culture room (per team):''' | '''in tissue culture room (per team):''' | ||
Line 43: | Line 43: | ||
**3.0 mL RW1 | **3.0 mL RW1 | ||
**4.2 mL RPE | **4.2 mL RPE | ||
− | **140 μL | + | **140 μL RNase-free H<sub>2</sub>O |
*cDNA aliquots | *cDNA aliquots | ||
Line 64: | Line 64: | ||
'''in main lab (per team):''' | '''in main lab (per team):''' | ||
− | * | + | *2.75 mL RNase-free H<sub>2</sub>O |
*qPCR aliquots | *qPCR aliquots | ||
− | ** | + | **420 μL Buffer PB |
+ | **1.5 mL Buffer PE | ||
+ | *30 μL GAPDH primer mix | ||
+ | *30 μL p21 primer mix | ||
==M2D6== | ==M2D6== | ||
Line 84: | Line 87: | ||
==M2D8== | ==M2D8== | ||
'''24 hr preceding laboratory:''' | '''24 hr preceding laboratory:''' | ||
− | *seed DLD-1 (6 wells at 25K) and BRCA2-/ | + | *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):''' | '''in tissue culture room (per team):''' |
Latest revision as of 15:47, 8 March 2018
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