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

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  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°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°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°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°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