Reducing Background from Double Digested Vector

Reducing Background from Double Digested Vector

Having trouble with the background number of colonies produced by a vector that you have digested with two enzymes? The cause could be contamination by singly digested vector plasmids that are very likely to religate. This procedure is designed to reduce the background by removing phosphates from molecules digested with the first enzyme. Digesting with the second enzyme follows.

1. Digest 2000 ng of vector with first enzyme in 40 ul, incubate 30 minutes 37 C
2. Add 4.5 ul 10X Antarctic Phosphatase buffer and 1 ul Antarctic Phosphatase and incubate 30 minutes, 37 C
3. Run agarose gel and purify vector fragment
4. Digest with second enzyme in 20 ul
5. Heat inactivate second enzyme, or purify fragment from gel
6. Quantitate and use in ligation