Hairpin template in a PCR
nobody at nowhere.com
Wed Sep 27 17:36:15 EST 2006
> I am trying a strange PCR designed to transform a short DNA fragment (500
> bp) into an inverted repeat. Basically the idea is to: 1) ligate the
> fragment to a hairpin oligo and 2) Set up a PCR with a single oligo that
> binds to the opposite end. I have put and image to make this clearer in
Hi...the complementary part of the hairpin is quite large, isn't it?
I would be surprised to see that you get anything with Taq. When you
think about it, a 50 mer duplex would melt at 80C or something like
that, so you really need to find some additive that will split the
duplex apart and allow a polymerase to plow through.
You could also try 1M betaine, or even single strand binding protein.
And add a whack load of primer. I think Invitrogen has some DNa binding
proteins in their Accuprime Taq. You could also try Phusion or
PfuUltraII to see if they will work since they have a built in DNA
binding domain. Vent is apparently a strong strand displacer at higher
temp and is less prone to slippage in the presence of secondary
structure (I forget the reference).
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