full length cDNA question

Caroline Szymeczek-Seay css at med.unc.edu
Fri Jan 7 17:06:44 EST 2000


I am trying to obtain a full-length clone by RT-PCR. We could not design
primers that would amplify the entire 1.9 kb fragment because of
sequence considerations (couldn't get 2 primers with similar enough
Tm's).  Therefore we designed 2 sets of primers.  Each set would amplify
a portion of the sequence.  The resulting products are 1082 bp and 1268
bp.  These two sequences share a 415 bp overlap.  My question is, how
can I generate a full length clone from these two overlapping PCR
products?  

I was considering just denaturing them in the same tube, and then using
the forward primer from one set of primers and the reverse primer from
the other set of primers to PCR up the whole clone.  However, the
primers won't be similar in Tm.  I'm guessing this may not be a big
problem since the template will be much more specific than the original
RNA to cDNA template.  Does anyone have any specific tips re: this
method?  Or any suggestions for other methods?  much thanks in advance..


caroline


Caroline Szymeczek-Seay
Research Assistant Professor
University of North Carolina at Chapel Hill
Chapel Hill, NC  27516-7220




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