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.
Spin 1.5ml o/n culture at 1,2000rpm for 30 Sec. Discard the supernatant.
Resuspend the pellet by vertex in 100ul QP buffer, R.T. 5min.
Mix 200ul 10N NaOH and 500ul 20% SDS add water to final volume 10ml.
Add 200ul the solution above to the suspension (#2), invert 5 times, R.T. 5min.
Add 150ul 3M KAc (pH5.5) to #4, invert 5 times, ice 5min.
Spin at 12,000rpm for 5min.
Remove 300ul supernatant to a new tube, add 350ul phenol/chloroform, vertex 15 sec, and centrifuge for 5min.
Remove 200ul supernatant from the top phase, add 500ul cold ethanol, -20oC for 30min.
Spin 12,000rpm 10min at R.T, and air dry for 5min.
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