20.109(F16): TA notes for M2

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