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

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)
• glass culture tubes (4)
• 15 mL conical tubes (4)

M2D8

On front bench, per team:

• cuvettes for spectrophotometer (9)
• conical tubes (4)
• eppendorf tubes (16)
• on ice: for kits
  • standard
  • substrate
  • enzyme
• aluminum foil

Aliquot, per team

• LB (20 mL for ethanol, 10 mL for lactate)
• PBS (none for ethanol, 4 mL for lactate)
• assay buffer (4.5 mL for ethanol, 4 mL for lactate)