I am cloning a PCR blunt product into pJET1.2 vector. The colony PCR and plasmid PCR have confirmed the presence of insert. The insert size is 1.17kb and the plasmid with insert looks 4kb on the gel with liberalized plasmid However, when I do digestion with HindIII and EcoRI the insert is not released. The individual digestion seems to linearize the plasmid but the double digestion does not release the insert (I tried both sequential and combined double digestion). I have not yet sequenced the plasmid.
Could be the insert has a second restriction site close to the other sort and one of the restriction sites you intend to cut has been mutated by a blunt end ligation.
Robert Adolf Brinzer We digest individually the digestion looks good
We have included EcoRI and HindIII in our primers so PCR product we ligated should carry these 2 sites Tom Masi
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