Difference between revisions of "20.109(F16): TA notes for M2"
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MAXINE JONAS (Talk | contribs) (→M2D5) |
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| Line 62: | Line 62: | ||
*PB(1100 μL) | *PB(1100 μL) | ||
*PE (1600 μL) of buffer PE | *PE (1600 μL) of buffer PE | ||
| − | * | + | *water pH 8.0 for elution (50 μL) |
| + | *2 mini-prep columns | ||
*MG1655 competent cells (100 μL), '''on ice''' late in the afternoon | *MG1655 competent cells (100 μL), '''on ice''' late in the afternoon | ||
| + | *SOC (1 mL) | ||
| + | *2 LB agar plates +Amp+Cam | ||
| + | *burner + lighter + spreader + jar of ethanol | ||
On front bench, | On front bench, | ||
| Line 69: | Line 73: | ||
*water bath at 42 °C | *water bath at 42 °C | ||
*sequencing F and R primers at 5 μM | *sequencing F and R primers at 5 μM | ||
| + | *nuclease-free water | ||
| + | *PCR tube strips for sequencing | ||
===M2D7=== | ===M2D7=== | ||
Revision as of 18:05, 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
- water pH 8.0 for elution (50 μL)
- 2 mini-prep columns
- MG1655 competent cells (100 μL), on ice late in the afternoon
- SOC (1 mL)
- 2 LB agar plates +Amp+Cam
- burner + lighter + spreader + jar of ethanol
On front bench,
- students plates of pgRNA
- water bath at 42 °C
- sequencing F and R primers at 5 μM
- nuclease-free water
- PCR tube strips for sequencing
M2D7
Aliquot, per team:
- LB broth (45 mL)
- chloramphenicol (50 μL, from stock in -20 °C freezer)
- ampicillin (50 μL, from stock in 4 °C deli fridge)
