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        Plasmid or Cosmid DNA Miniprep

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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, -20o C 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
        • 37o C 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 4o C.
        Phenol/chloroform:
        • 25ml buffer equilibrated phenol (Sigma)
        • 24ml chloroform
        • 1ml isoamyl alcohol

         

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