20.109(F19): Prep notes for M2
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M2D1
per team:
- 100 μL nuclease-free H2O2
- ~ 2 μg pdCas9
- not aliquoted: enzyme stocks, buffer stocks
M2D2
per team:
- 25 μL 6X loading dye
- not aliquoted: 1 kb ladder stock, electrophoresis buffer
M2D3
per team:
- 750 μL nuclease-free H2O2
- not aliquoted: nuclease-free H2O2, pgRNA vector, gRNA reverse primer, Q5 Master Mix
M2D4
per team:
- none
M2D5
per team:
From Qiagen mini-prep kit
- 550 μL buffer P1
- 550 μL buffer P2
- 750 μL buffer N3
- 1 mL buffer PB
- 1.6 mL buffer PE
- 70 μL nuclease-free H2O2, pH = 8
- 1 mL SOC
not aliquoted: gRNA sequencing primers
M2D6
per team:
- none
M2D7
per team:
- 45 mL LB broth
- 50 μL chloramphenicol stock
- 50 μL ampicillin stock
M2D8
per team:
- each team uses either Ethanol kit OR Acetate kit reagents
From Ethanol kit (store at 4°C, except ethanol probe at -20°C)
- 20 mL LB broth
- 5 mL ethanol assay buffer
- 83 μL ethanol probe
- 83 μL ethanol enzyme
From Acetate kit (store at -20°C, except LB at 4°C)
- 10 mL LB broth
- 5.6 mL acetate assay buffer
- 86.4 μL acetate probe
- 86.4 μL acetate substrate
- 86.4 μL ATP
- 86.4 μL acetate enzyme