multiple bands on 2nd rd PCR

Jeff Lawrence lawrence at bioscience.utah.edu
Tue Jun 13 11:33:31 EST 1995


In article <3ri9u4$as6 at mark.ucdavis.edu>, ez022056 at dale.ucdavis.edu
(Edward Wang) wrote:

> I am PCRing bands that were 
> extracted from acrylamide gel.  Some of them show up with the first round 
> of PCR.  Others require a second round of PCR.  However, after the second 
> round, some of my products had multiple bands (2-4).  Does anybody have 
> any explaination for this other then contamination?

we have had similar experiences, and some of those bands may be
amplification products nested within your original band (assuming they're
smaller :-)).  we have sequenced a few and found this to be the case. 
this results from the high amount of DNA used as template. to avoid them,
reamplify using reaction conditions more stringent than your primary
amplification by increasing anneal temps, reducing elongation times, and
decreasing magnesium concentrations.  these have all worked for me.

jeff

-- 
jeff lawrence
lawrence at bioscience.utah.edu



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