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)

• razor blades (1)
• bottomless 96-well plates (3)
• glass slides (2)
• binder clips (8)
• water bath at 43 °C with
• 1% LMP agarose (3 mL)
• 12-well reservoirs (1)
• multi-channel 300 μL pipets (1)
• boxes for lysis incubation (1 dead, 1 live)
• lysis buffer stock (40 mL)
• Triton X-100 (400 μL)
• Neutralization buffer (50 mL)
• SYBR Gold buffer (40 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, on front bench, prepare (per group)

• M and L flocked-lined gloves
• MMS at 80 mM (1.25 mL RPMI + 8.5 μL of 12 M stock, prepared in fume hood)
• RPMI1640 medium (12 mL)
• PBS (20 mL)
• dedicated MMS liquid waste basins

For MMS, aliquot (per team)

• TK6 media
• MMS at 80 mM (250 μL)

M1D4

In incubator, have (per group)

• Coriell # 10 (300,000 cells)
• Coriell # 20 (300,000 cells)
• Coriell # 24 (300,000 cells)

Per group, aliquot:

• 25 mL 1x PBS
• 25 mL RPMI 1640 media (non sterile, warm in incubator)
• liquid trash basin

On front bench, prepare (per team)

• razor blades (1)
• glass slides (1)
• binder clips (4)
• bottomless 96-well plate (1)
• 1% LMP agarose (2 mL)
• water bath at 43 °C
• cold H2O2 at 10 mM (10 mL PBS + 10 &muL H2O2 10 M stock; each team needs < 100 μL)
• reservoir (1)
• multichannel pipet (1)
• tray boxes (1 + 1 for lysis and for recovery)
• lysis stock buffer (30 mL)
• Triton X-100 (300 μL)

Tomorrow, will need (per team)

• Neutralization buffer (75 mL)
• SYBR Gold buffer (40 mL)
• electrophoresis gel boxes with double-sided tape (2 teams or 2 CometChips per box)
• alkyline electrophoresis buffer (500 mL)

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:

• 5 mL TBS
• 5 mL blocking buffer (4.5 mL of TBS + 500 μL of 10% BSA, frozen stock in -20C)
• 700 μL blocking buffer
• one staining chamber
• one gauge 26 needle
• one pair of tweezers

On front bench, prepare:

• Triton X-100
• Primary antibodies

M1D7

Per group, aliquot

• 1.5 mL of 1x TBS
• 4 microscope slides
• 1 gauge 26 needle

On front bench, prepare

• ProLong Gold anti-fade reagent with DAPI
• beaker of water
• nail polish
• book to transport slides