Can I increase RT stringency?

Darby Crash? phelan at jsei.ucla.edu
Thu Sep 28 14:17:35 EST 1995


Hello all,

I have been using RT-PCR to amplify a member of a group of closely
related sequences.  The system is such that the forward primer 
probably exactly matches more than one of these sequences, and 
so I need to rely on the reverse primer to generate the specificity.  
Unfortunately, I end up with bands from more than one sequence.  
I think this is primarily due to mispriming at the RT step.  
For my use, I really want only one band.  Therefore, 
my question is this:

Is there a way to increase the stringency during RT with MuLV to 
prevent mispriming?  I would prefer to make adjustments without 
switching enzyme systems.

Please reply by e-mail, as I can't always reach the news group.
Thanks,
DC?



More information about the Methods mailing list