NESTED PCR of a nested PCR, necessary?

Ray Lara rlara at nmu.edu
Sat Jun 5 11:06:03 EST 1999


I have a query about nested PCR...

I want to sequence a PCR fragment that
I obtained via nested PCR.  My primary PCR comes up with multiple smeary
fragments from a Vectorette PCR (definitely not routine PCR!).  Then I took
1ul of reaction and ran it in a semi-nested PCR (one primer nested) and got
a very bright band, though this band was not present in the initial
PCR.

My question:  Intuitively, all statistics are in my favor for being
the fragment that I want, correct?  Do you think it is necessary to do a
nested PCR of a nested PCR to get the SPECIFIC band of interest?  

The
problem with Vectorette PCR is that the size of the fragment is not really
known, it is dependent on the restriction sites that are part of the
protocol.

If anyone has any suggestions or comments, please write.

Thanx
in advance

Jonathan



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