Swartz:Protocols/Maxiprep: Difference between revisions
m (New page: 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 ...) |
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Revision as of 12:33, 5 May 2012
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