Difference between revisions of "20.109(F16): TA notes for M1"

M1D1

Per group, aliquot

• 50 mL 1x PBS
• in TC room, 15 mL of “TK6 media”

M1D2

Per group, aliquot

• in main lab
  • 10 mL 1x PBS
  • 2 eppendorf tubes of 0.3% LMP agarose (in eppendorf water bath)
  • large scheme of the plate so they can keep track of how to load chips
• and separately, in the TC hood,
  • 10 mL 1x PBS
  • 25 mL TK6 media
  • 2 eppendorf tubes of 0.3% LMP agarose (in water bath at 43 °C)
  • 2x 15 mL of TK6 cells at 500,000 cells/mL

Set up hoods in TC room with, per group:

• 4 binder clips
• 1 glass slide
• 1 bottomless 96-well plate
• 1 razor blade
• Pasteur pipets
• sharp jar
• pipet aid
• pen
• team sticker to identify glass slides
• printout of today's protocol
• large scheme of the plate to hang in TC so they can keep track of how to load chips

M1D3

In incubator, have (per group)

• TK6 cells at 500,000 cells/mL (10 mL)

On front bench, prepare (per group)

• bottomless 96-well plates (3)
• glass slides (2)
• binder clips (8)
• water bath at 43 °C with
• 1% LMP agarose (3 mL)
• lysis buffer stock (40 mL)
• Triton X-100 (400 μL)
• Neutralization buffer (50 mL)
• SYBR Gold buffer (20 mL)

In cold room, prepare (per group)

• electrophoresis gel boxes with double-sided tape (2 teams or 4 CometChips per box)
• alkyline electrophoresis buffer (500 mL)

For H2O2, on each team's bench, prepare

• ice bucket with
• 12-well reservoir
• 50 mL PBS
• H2O2 at 10 mM in cold PBS

For MMS, aliquot (per team)

• TK6 media
• MMS at 80 mM (250 μL)

M1D4

Per group, aliquot:

• 15 mL 1x PBS
• H2O2
• Alkyline lysis solution
• Warm M059J/K culture medium

M1D5

Per group, aliquot in TC room:

• 5 mL of 0.1% gelatin
• 10 mL of PBS
• 3 mL of 1X trypsin
• 15 mL of M059J/K media
• M059J/K in T25 flasks

M1D6

Per group, aliquot in TC room:

• 10 mL of 1x TBS
• 500 uL of 10% BSA, frozen stock in -20C
• Triton X-100 at front bench
• Primary antibodies

M1D7

Per group, aliquot

• 5 mL of 1x TBS