Meaning of high MW PCr smears

Steven Sullivan sullivan at
Mon Jun 2 23:03:27 EST 1997

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? 

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