Difference between revisions of "20.109(F16): TA notes for M2"
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*NEB5α cells (1 per group + 1 for control) | *NEB5α cells (1 per group + 1 for control) | ||
*LB+Amp plates (1 per group + 1 for control) | *LB+Amp plates (1 per group + 1 for control) | ||
+ | |||
+ | ===M2D5=== | ||
+ | The day before lab (8pm), inoculate 2 colonies per team. | ||
+ | |||
+ | Aliquot, per team: | ||
+ | *2x 5 mL cultures of NEB5α transformed with pgRNA and cultured in LB+Amp | ||
+ | *ice bucket | ||
+ | *15 mL conical tube, empty, labeled 'Qiagen waste' | ||
+ | *P1 (600 μL) | ||
+ | *P2 (600 μL) | ||
+ | *P3 (800 μL) | ||
+ | *PB(1100 μL) | ||
+ | *PE (1600 μL) of buffer PE | ||
+ | *waster pH 8.0 for elution (50 μL) | ||
+ | *MG1655 competent cells (100 μL), '''on ice''' late in the afternoon | ||
+ | |||
+ | |||
+ | On front bench, | ||
+ | *students plates of pgRNA | ||
+ | *water bath at 42 °C | ||
+ | *sequencing F and R primers at 5 μM | ||
+ | * |
Revision as of 17:04, 27 October 2016
M2D1
Per group, aliquot
- 30 μL nuclease-free water
- 5 μL pdCas9 (ideally at 500 ng/μL)
On front bench, have
- restriction enzymes in -20 °C block
- NEB buffers
- list of 20.109-owned restriction enzymes
Fa16: all done by 5pm
M2D2
Per group,
- pour 1/2 agarose gel (only 5 lanes needed)
- thaw 4 samples of confirmation digests
- aliquot 10 μL of 6X loading buffer
- thaw 20 μL of 1 kb ladder
Call IDT to request expedited FedEx shipping of gRNA oligos.
Fa16: all done by 4:30pm Should schedule BE Comm Lab workshop on Journal Club on this day.
M2D3
On ice,
- 1.2 mL aliquots of nuclease-free water
- reverse primer at 100 μM
- psgRNA template
- HotStart Master Mix
- -20 °C block for PCR tubes
- strip of PCR tubes with colors (+1 for control)
- SDM control primer mix
- SDM control plasmid
Also on front bench,
- filtered tips
- gRNA forward primers received from IDT
The thermocycling program is called NEBSDM.
Later, on ice
- KLD reaction buffer
- KLD enzyme mix
- NEB5α cells (1 per group + 1 for control)
- LB+Amp plates (1 per group + 1 for control)
M2D5
The day before lab (8pm), inoculate 2 colonies per team.
Aliquot, per team:
- 2x 5 mL cultures of NEB5α transformed with pgRNA and cultured in LB+Amp
- ice bucket
- 15 mL conical tube, empty, labeled 'Qiagen waste'
- P1 (600 μL)
- P2 (600 μL)
- P3 (800 μL)
- PB(1100 μL)
- PE (1600 μL) of buffer PE
- waster pH 8.0 for elution (50 μL)
- MG1655 competent cells (100 μL), on ice late in the afternoon
On front bench,
- students plates of pgRNA
- water bath at 42 °C
- sequencing F and R primers at 5 μM