Meaning of high MW PCr smears

s.pritchard mmd280 at sysc.abdn.ac.uk
Thu Jun 5 05:02:51 EST 1997


Steven Sullivan (sullivan at gwis2.circ.gwu.edu) wrote:


: I'm trying to re-amp some PCR products from a gel -- running them in an
: EDTA-free gel and buffer, taking a plug, melting it in 20X volume of
: distilled water (95 degrees for a few minutes; the gel is *not* low melt),
: using 1 ul in a 50 ul PCR reaction, with M13 F and R primers at 55 degree
: anneal temp (same as original PCR). ANd I keep getting smears starting
: from around the expected product size up to several kb higher.  No
: primer-dimers in evidence (I'm doing hotstarts).  Am I adding too much
: DNA, or is there some other parameter that needs changing? 



I have seen this aswell ......I think that it could be caused by too much
template DNA as this can cause non specific binding of the primers.Try dilutions
of your template.
Also you could check that your Taq is still 100% active as that could cause
similar problems.

Hope this is of some help and good luck

Stuart Pritchard



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