3kb PCR amplification

Patrick Lynch
Thu Mar 15 12:33:15 EST 2001


A lot depends on the quality of your RNA and the sequence of the
transcript.  Good quality = long transcripts, bad quality = short
transcripts, high GC content = short transcripts (generally).  Also, you can
purchase specialised RT kits that claim to give longer cDNAs - check out the
range from Stratagene.  From my own experience, I've managed to amplify
different 2 kb PCR products from first-strand cDNA occasionally and 1 kb
PCRs regularly.  3.7 kb sounds a bit too ambitious for RT-PCR.  Still, if I
were you, try to amplify the 3.7 kb fragment first - use a good quality Taq
that is known to work with difficult PCRs and plenty of PCR conditions.  If
this fails, then try amplify the sequence in smaller fragments and "stitch"
the fragments together by cloning (or some other means).

Hope this helps,

Patrick


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