PlasmidorCosmidDNAMiniprep

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, -20^oC 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

• 37^oC 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 4^oC.

Phenol/chloroform:

• 25ml buffer equilibrated phenol (Sigma)

• 24ml chloroform

• 1ml isoamyl alcohol