Difference between revisions of "20.109(F16): TA notes for M1"
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MAXINE JONAS (Talk | contribs) (→M1D3) |
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| Line 37: | Line 37: | ||
On front bench, prepare (per group) | On front bench, prepare (per group) | ||
| + | *razor blades (1) | ||
*bottomless 96-well plates (3) | *bottomless 96-well plates (3) | ||
*glass slides (2) | *glass slides (2) | ||
| Line 42: | Line 43: | ||
*water bath at 43 °C with | *water bath at 43 °C with | ||
*1% LMP agarose (3 mL) | *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) | *lysis buffer stock (40 mL) | ||
*Triton X-100 (400 μL) | *Triton X-100 (400 μL) | ||
*Neutralization buffer (50 mL) | *Neutralization buffer (50 mL) | ||
| − | *SYBR Gold buffer ( | + | *SYBR Gold buffer (40 mL) |
In cold room, prepare (per group) | In cold room, prepare (per group) | ||
| Line 56: | Line 60: | ||
*50 mL PBS | *50 mL PBS | ||
*H2O2 at 10 mM in cold 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) | For MMS, aliquot (per team) | ||
Revision as of 15:27, 21 September 2016
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
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
