Swartz:Protocols/Maxiprep

From OpenWetWare

< Swartz:Protocols
Revision as of 18:46, 28 May 2012 by Christopher C Vanlang (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

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
Personal tools