Difficult RT-PCR

Jan-Henner Wurmbach wurmbach at uke.uni-hamburg.de
Wed Oct 27 03:31:08 EST 1999



Andrea Gilpin <gilpin at botany.utoronto.ca> schrieb im Beitrag
<38161183.1FB2C604 at botany.utoronto.ca>...
> Dear Everyone:
> 
> We are currently facing difficulty getting RT-PCR products.  Our gene
> has a genomic length of 9 kb, in which there are 8 exons making the
> total coding sequence of 3.6 kb.  We can easily get a 1.2 kb cDNA
> spanning exons 1 and 2, and recently, we have also obtained a 2.9 kb
> fragment of cDNA from exon 1 to exon 6.  But we have had tremendous
> difficulty getting the full-length cDNA with the RT-PCR method.  Does
> anyone have any suggestions, recommendations and/or tips.
> 
> Thanks very much,
> 
> Zhou
> zhou at botany.utoronto.ca

Might your problem be the RNA or the RT?
Bad RNA means no good RT and only short cDNAs.
Also, how do you make your First Strand? Random primer, Oligo-dT or
specific?
If you random prime you probably wont get long Fragments.

Hope these short suggestions helped
   Jan-Henner Wurmbach  <wurmbach at uke.uni-hamburg.de>



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