Sequencing Gels

Reid, Thomas M. tar9 at NIOBBS1.EM.CDC.GOV
Fri Mar 10 12:38:15 EST 1995


Hi-
I am trying to sequence an 800 bp cDNA amplified by PCR.  I am using 
end-labeled primers and Sequenase (ver 2.0) enzyme.  I have 2 problems that 
I would appreciate any comment on.

I am getting strong termination bands approximately 250 bases away from the 
primer.  This happens with all three primers I am using so it is not 
sequence-specific.  My termination reactions contain a 20:1 dNTP to ddNTP 
ratio.  Is there any way to increase the length of DNA that is polymerized 
by Sequenase?

This is probably a trivial problem but sometimes they can be the most 
frustrating.  I am getting fairly good resolution of bands near the bottom 
of gels and especially near the top of my gels.  However in the middle of 
some (but not all) gels the bands are smeared quite a bit and are almost 
impossible to read.  I suspect some kind of gel artifact but am not sure 
where to start.

Thanks for any help - TR



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