20.109(S18): Prep notes for M2

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20.109(S18): Laboratory Fundamentals of Biological Engineering

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Spring 2018 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Assessing ligand binding        2. Measuring gene expression        3. Engineering biomaterials              


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