2nd round of PCR

Bruce Byrne byrne at soma.umdnj.edu
Tue Aug 18 08:22:00 EST 1992


walterh at U.WASHINGTON.EDU (Walter Hill) writes:

>Dear fellow net-rats:
>A colleague of mine is amplifying rotavirus gene using RT-PCR and that works
>well and a second round of PCR cycles using nested primers also works well.
>However, when the second amplification is performed with primer identical to
>the first amplifcation, no or very little amplification occurs.
>Can anyone provide an explaination for this phenomenon?

Maybe.  If the original target were rare or not a very good fit to your 
primers, your colleague likely formed some primer dimers.  Those unwanted
PCR products are perfect targets for the second round of amplification by
the same primer pair.

Added note of caution.  Any second round of amplification adds great
complexity to handling carryover contamination.  

Cheers!

Bruce Byrne



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