Difference between revisions of "20.109(F16): TA notes for M1"
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+ | {{Template:20.109(F16)}} | ||
− | + | <div style="padding: 10px; width: 790px; border: 5px solid #000FFF;"> | |
− | + | ===M1D1=== | |
− | + | Per group, aliquot | |
+ | *50 mL 1x PBS | ||
+ | *in TC room, 15 mL of “TK6 media” | ||
− | + | ===M1D2=== | |
− | + | Per group, aliquot | |
− | Per group | + | *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: | + | 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 |
Latest revision as of 13:14, 6 October 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
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