Hairpin template in a PCR

Austin So nobody at nowhere.com
Wed Sep 27 17:36:15 EST 2006


TR wrote:

> 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
> http://www.flypicture.com/bin/?id=qNj0karb

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).

Cheers

Austin



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