reducing secondary structures in long PCR primers
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Fri Jun 16 04:11:27 EST 2000
In article <8ibk48$n2l$1 at oyez.ccc.nottingham.ac.uk>, Benny Lo
<benny at holmes.nott.ac.uk> writes
>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.
What happens when you put your primer sequence against say Mfold. Do you
have such loops?
Alternatives are that you received poorly synthesised primer with no
Hind III site in or that you have a nuclease in your ligation that is
chewing up the ends of the insert.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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