Methods Digest, Vol 56, Issue 10 (Message 2:Joining two PCR products together)

Peter Ellis via methods%40net.bio.net (by pjie2 from cam.ac.uk)
Tue Jan 12 04:54:21 EST 2010


Liyana Ismail wrote:
> Hi Catherine,
> 
> I'm tempted to reply to this one since we're doing something pretty similar.

Yup, this should be a workable protocol in the general case.  The 
problem here is that Catherine only has 10-15bp of overlap between the 
two original products, as compared to your 319bp.  That will make the 
joining reaction much more difficult, since you can't do it at PCR 
temperatures.

To be honest, with that small a degree of overlap, Catherine might be 
better placed incorporating appropriate restriction sites into her 
primers and then cutting/ligating.  It depends on how critical the exact 
sequence of the joining region is and whether she has the leeway to 
design in a RE site.

Peter


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