I will be running inverse PCR to sequence the segments adjacent to my target segment. One of the template sources will be very difficult obtain again. I first ran the non-inverse PCR to discover which samples contained the segment. Can I use this PCR product as the template for the inverse PCR (using inverse sequences of the former primers)? The original template should still be there, right? In fact, when I ran the gel to verify the presence of my segment, I gel-purified all DNA >2000 bp, which should have the original template minus the amplified segment and primers. If I use this DNA as template for inverse PCR, is there any special protocol I need to follow?
Hi there,
If the DNA template is circular (continuity between primers), inverse PCR should be OK if the template has efficiently been purifed from the regular PCR reaction.
I will repeat what Dominique mentioned, plus I will add that with that: amplicon you cant do inverse PCR, it can be only done in a circular plasmid
and if you already have pcr amplified material it will be easy to add a restriction site to the primers and re amplify more material and make ligation easier
My template is linear not circular, but I will circularize it using restriction enzymes and ligase.
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