Reverse Transcription

denton at usuhs.ucc.usuhs.nnmc.navy.mil denton at usuhs.ucc.usuhs.nnmc.navy.mil
Wed Dec 22 15:36:56 EST 1993


In Article <2f9ooh$bf4 at serss0.fiu.edu>
harkinsd at solix.fiu.edu (Derek M. Harkins) writes:
>VAN BELZEN @ PA1 (belzen at pa1.fgg.eur.nl) wrote:
>: In article <2f5bu4$ah1 at post.its.mcw.edu> abuhajir at post.its.mcw.edu (Majed Abu-Hajir) writes:
>
>: >Any one on the net had experience with rTth DNA polymerase?
>  It did not work for me.
>"classical" methods to successfully cDNA and amplify my RNA samples.
>Using the same samples and primers, I couldn't get the PE kit to work.
>I dont know--maybe I just got a "bad" kit.  BTW  I use M-MLV RTase in my applications. 
    I too have tried using this enzyme kit to analyze 5'ends in an extra-
ordinarilly GC rich region of mRNA.  The results were abyssmal.  My 
confidence is still with good old fashioned MMLV RT, which has, for all
of its' shortcomings, given reproducible, (but not always intelligible!)
results.  I gave up on the rTTh after the first try, so it is possible
that some tweaking might make it work.... if you like trouble-shooting
experimental procedures.  My guess is that the generic conditions that
PE prescribes for the use of the enzyme are geared for subsequent use
of the product in PCR amplifications, and only represent "garden variety"
primer extensions.  If somebody has conditions for making this work for
the analysis of mRNA, I would be very interested in hearing from you.
    R. Rex  Denton, Ph. D.
    Denton at USUHSB.USUHS.NNMC.NAVY.MIL



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