Swartz:Protocols/Maxiprep
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https://docs.google.com/document/d/1woT8L647962azzQERKb_4_GdmsoBTWKpRtyVVSj2OTE/edit
Qiagen DNA plasmid MaxiPrep
- Day 1:
- Inoculate 5 mL of selective LB broth from glycerol stock or plate colony
- Grow at 37 °C, ~8 h with shaking
- Autoclave 1L LB media
- When cooled, add appropriate antibiotic
- Add entire contents of 5mL starter culture to 1L media
- Grow at 37 °C, 12-16 h overnight with shaking
- Day 2:
- Dispense culture in 1L bottles and harvest in centrifuge at 6,500 rpm, 30 min
- While culture is spinning, put buffer P3 on ice
- Resuspend the pellet with 40 mL of buffer P1 using the hand homogenizer
- Add 40 mL of buffer P2
- Mix thoroughly by inerting vigourously
- Incubate at room temperature for no longer than 5 min
- Add 40 mL of chilled buffer P3
- Incubate on ice, 20 min
- Spin down lysates at ~20,000g, 30 min, 4 °C
- While spinning, prepare 2 Qiagen-500 tips per L of culture by adding 10 mL buffer QBT
- Remove supernatant into clean bottle by pipette
- Filter through Qiagen filter (0.2-0.4 µm filters can also be used)
- Load on columns by gravity flow
- Wash columns 2x with 30 mL Wash Buffer
- While loading and washing columns, place 25 mL isopropanol per L of culture in -30 °C freezer
- Elute with 15 mL buffer QF into clean 50 mL Falcon tube
- Precipitate DNA with 10.5 mL cold isopropanol
- Centrifuge at ~20,000g, 30 min, 4 °C
- Pour of supernatant
- Be careful not to disturb DNA pellet
- Wash the pellet with 0.5 mL of 70% ethanol
- Transfer to 1.5 mL Eppendorf with transfer pipettes
- Centrifuge at 12,000 rpm, 20 min
- Pipette off ethanol
- Allow pellet to dry either overnight on benchtop or 30-45 min in centrivap
- Resuspend pellet in 100-200 µl water
- Flick tubes to speed up resuspension
- Allow to resuspend on benchtop for several hours or overnight
- Check concentrations on spectrophotometer by measuring absorbance at 260 and 280 nm