DDRT-PCR

Mike Rott mrott at unixg.ubc.ca
Fri Apr 29 13:00:55 EST 1994


I have been trying the DDRT-PCR technique with inconsistant results. 
Sometimes I get a very nice signal with lots of bands but in the next run
nothing, although I use the same RNA source and primers. I have found that
different sources of Taq work better than others. Perkin-Elmer Taq seems to
work better for me but I need to use 2 units instead of 1 per reaction.  I
use the GIT method to extract my RNA and then treat with GIBCO DNAase.  I
get an A260/280 of greater than 1.8 and on an agarose gel I get a nice
smear with sharp ribosomal RNA bands and no high MW bands, so I think my
RNA is OK. I also get good first strand synthesis as indicated by TCA ppt
and gel electrophoresis.  I don't have much PCR experience and haven't yet
played around too much with the PCR reaction conditions other than Taq and
cDNA concentrations.

Any suggestions would be appreciated, 

In addition, are people using denaturing or non-denaturing gels?  I perfer
the banding pattern of the nondenaturing gels but the lanes don't run as
straight as a denaturing gel.

thanks,    Mike   



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