IGEM:IMPERIAL/2007/Notebook/General Protocols/Midiprep


 * 1) Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2-5ml LB medium containing the appropriate selective antibiotic. Incubate for approx. 8h at 37&deg;C with vigorous shaking (~300 rpm)
 * 2) Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy plasmids inoculate 50ml medium. For low-copy plasmids, inoculate 150ml medium. Grow at 37&deg;C for 12-16h with vigorous shaking (~300 rpm)
 * 3) Harvest the bacterial cells by centrifugation at 60000 x g for 15 min at 4&deg;C
 * 4) Resuspend the bacterial pellet in 6ml buffer P1
 * 5) Add 6ml buffer P2, mix thoroghly by vigorously inverting the sealed tube 4-6 times, and incubate at room temperature (15-25&deg;C)for 5 min
 * 6) Add 6ml chilled buffer P3 to the lysate, and mix immediately and thoroughly by vigorously inverting 4-6 times. Do not incubate the lysate on ice
 * 7) Pour the lysate into the barrel of the QIAfilter Catridge. Incubate at room temperature for 10 min. Do not insert the plunger
 * 8) Equilibrate a HiSpeed Midi Tip by applying 4ml buffer QBT and alow the column to empty by gravity flow
 * 9) Remove the cap from the QIAfilter outlet nozzle. Gently insert the plunger into the QIAfilter Midi catridge and filter the cell lysate into the previously equilibrated HiSpeed Tip
 * 10) Allow the cleared lysate to enter the resin by gravity flow
 * 11) Wash the HiSpeed Midi Tip with 20ml buffer QC
 * 12) Elute DNA with 5ml buffer QF
 * 13) Precipitate DNA by adding 3.5ml room temperature isopropanol to the eluted DNA. Mix and incubate at room temperature for 5 min