DNA Reaction - Inverse PCR

Jeff Lawrence

1. Obtain primers that direct replication away from each other. Optimally, primers are about 200 bp from the end of known sequences

2. Cleave DNA with a six-base recognition-site restriction endonuclease that does NOT cleave between the two primers of interest. Alternatively, perform a partial digestion with a 4-base recognition-site enzyme.

3. Purify the DNA after digestion. DO NOT phosphatase.

4. Perform a ligation using a final DNA concentration of 2 fmol/10 mL, or 0.0002 µM. The low concentration selects for the formation of monomeric circles.

5. Purify and concentrate the DNA by ethanol precipitation. If prepared by partial digestion (and is generally applicable), this template can be stored for future use.

6. Perform the PCR with the two primers selected above using the purified, ligated DNA as template. The product will contain the sequences flanking the two primers:

Last Update: Thursday, 19-Jun-2014 11:50:16 PDT
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Eric Kofoid eckofoid at ucdavis.edu