I'm working on the cloning vector pET-15B, and I'm cleaving at the NdeI and XhoI restriction sites, and the only conceivable way I can think of is to run the product on a 5% agarose gel, which, if you've ever tried to make, is a pain in rump!
I suppose it depends on what you mean by completely but running 2 pcrs with primers including the cut out fragment and both failing to work would indicate that the sequence was missing from the vector. Or possibly cutting the cut product with a third enzyme giving a fragment where N and N-7 are easily distinguishable,, Does it make much difference to your next step if there is a little single cut product?
Thanks for your answer, Paul! Let me give you more background: I'm using random mutagenesis PCR to induce changes in the RFP gene, and then am cutting out the RFP from the pRFP I've solated, and then I'm putting the RFP into the pET-15b cloning vector. Its easy to see that my digests of the pRFP were successful, because I have 2 sets of bands (the RFP gene is about 680 bp.) but when I run a gel of the pET-15B digest, I only get one band because the cut in the vector is so small. Does that help explain what I'm doing?
Hi Angie,
I may be missing something but if you have checked that both enzymes cut in the buffer used or have done separate digests is it a reasonable supposition that the enzymes have cut and that cloning your fragment into the vector is as good a test as any that the digest was complete which is where you want to be as well
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