HELP -- pcr mystery

Juliewoods juliewoods at aol.com
Mon Jun 17 12:15:09 EST 1996


We have a strange result in our lab.  Usually we gel purify pcr products
for sequencing by removing a plug of the appropriate band from a lmp gel
with a glass pastuer pipette tip, melting in 200ul H2O, and using this as
a template for a second amplification at higher stringency.  It works
great -- supplies primer-ready template, gets rid of spurious bands, gives
bright crisp products.  

Recently, however, we have a set of samples that won't reamplify.  Well,
there is some activity but the re-amp products are worse (faint or streaky
or absent) than the initial amplifications.  We've lowered the annealling
temp. and added some Mg, but to no avail.  This doesn't make sense to us. 
Any ideas about what might be going on?  Please respond to: 
jfstein at AMNH.org        Thanks, julie



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