Swartz:Protocols/Maxiprep

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