20.109(F16): TA notes for M2
From Course Wiki
Revision as of 17:49, 20 October 2016 by MAXINE JONAS (Talk | contribs)
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)