Inverse PCR primers design - (Nov/30/2006 )
I have a 900 bp known fragment of an ORF and I want to perform an inverse PCR to sequence the flanking regions both, upstream and downstream. First I used a software to design regular primers towards internal sequences of the 900 bp, and for Inverse PCR I simply made the reverse complementary sequence of each primer, obtaining the outfacing ones. Is this approach ok?, Is there any tip you can give me?.
Also, I was planing to perform a gene library and look for the flanking sequences with the primers mentioned and primers of the cloning vector
Given that the 3' end of primers is most important, I would instead divide the fragment into a left and right half, then make a new fragment in order <right><left>. Then, I would use Primer3 to find primers. The real PCR will have an insertion at the site of the junction between the left and right fragments.
nice observation, THANKS!
hi phage 434.....
just one thing, is a bit stupid i know, but i want to be sure: creating in silico the molecule <right><left>, will give me outfacing primers without the need of changing to complementary -reverse sequences.... am I right?
Yes. Treat it just as you would in designing any other primer pair for a doing a PCR reaction on that region.