reducing secondary structures in long PCR primers

Benny Lo benny at holmes.nott.ac.uk
Thu Jun 15 17:13:13 EST 2000


Dear All

I have recently attempted adding a restriction sequence 5' to the gene I
have constructed by PCR. However, I found that the sequence (AAGCTT)
"disappeared" when I sequenced the resulting clones. The sequence (as part
of the primer) 5' of AAGCTT was there in the sequence, but when it reached
the point just before the AAGCTT sequence, it immediately went to the strat
of the gene, skipping the whole restriction sequence.

I 've repeated this set of PCR with an annealing temp of 64 deg., Taq
polymerase 15mM MgCl2 and I have the same results every time.

What I suspect here is that the restriction sequence was "looping out" in
the primer during the PCR process so the Taq enzyme could not extend
properly at that position.

Can anyone tell me if there is anything I can do to reduce such a problem?
Will it help if I use Pfx polymerase? Adding other substances to the PCR
mixture?

Thanks for any advice.









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