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Plasmid or Cosmid DNA Miniprep

来源:本站原创  2006/7/28 16:16:00   访问量:3114
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Plasmid or Cosmid DNA Miniprep


This protocol can be used to isolate sufficient amount DNA from 1.5ml o/n culture or 3ml 6hr culture to do several enzyme digestions.

  1. Spin 1.5ml o/n culture at 1,2000rpm for 30 Sec. Discard the supernatant.
  2. Resuspend the pellet by vertex in 100ul QP buffer, R.T. 5min.
  3. Mix 200ul 10N NaOH and 500ul 20% SDS add water to final volume 10ml.
  4. Add 200ul the solution above to the suspension (#2), invert 5 times, R.T. 5min.
  5. Add 150ul 3M KAc (pH5.5) to #4, invert 5 times, ice 5min.
  6. Spin at 12,000rpm for 5min.
  7. Remove 300ul supernatant to a new tube, add 350ul phenol/chloroform, vertex 15 sec, and centrifuge for 5min.
  8. Remove 200ul supernatant from the top phase, add 500ul cold ethanol, -20oC for 30min.
  9. Spin 12,000rpm 10min at R.T, and air dry for 5min.
  10. Resuspend in 35ul TE buffer with 1ug/ml RNase.
Digestion:
  • 10ul miniprep DNAl
  • 5ul restriction buffer
  • 1ul estriction enzyme (10U)
  • 24ul Water
  • 37oC for 2 hrs to overnight.
QP buffer:
  • 50mM glucose
  • 1ml 0.5M Tris-HCl (pH8.0)
  • 5ml 0.5M EDTA (pH8.0)
  • Add water to 200ml, sterilize by filtration and stored at 4oC.
Phenol/chloroform:
  • 25ml buffer equilibrated phenol (Sigma)
  • 24ml chloroform
  • 1ml isoamyl alcohol

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