sequencing ssDNA PCR

Marion D. Emerson mde5 at psu.edu
Mon Aug 21 12:33:30 EST 1995


Hello,

In our lab we routinely sequence ssDNA PCR products from 
mitochondrial DNA using the PCR primers in the sequencing
reactions.  This has worked well for us.
At this time I am trying to sequence ssDNA PCR products 
generated from RT-PCR of mRNA. Although I get great looking bands 
in both the RT-PCR reaction and the ss PCR reaction, when
I sequence (using the PCR primers), I get varying results.
One of the primers (5' to 3') works okay but the one in the 
other direction doesn't.  I get shadowy bands with lots of
backround, almost like a stuttering effect.  
Has anyone else had this problem and (hopefully) found a 
solution? 

Thanks in advance,    Marion

P.S.  we use a Taq sequencing method



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