Meaning of high MW PCr smears

[Brett Beitzel]

I've also noticed high MW smears on some of my PCRs.  They seem to appear
when I use long extension times (around 2 min.)  I don't see them at
shorter extension times.  Not really sure what they are.

Hope this helps,
Brett Beitzel
bbeitzel at jeeves.ucsd.edu


In article <5n052f$rkl$8 at cronkite.seas.gwu.edu>,
sullivan at gwis2.circ.gwu.edu (Steven Sullivan) 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?