St George's Hosp Medical School
fenechc at sghms.ac.uk
Wed Jan 11 12:21:17 EST 1995
i am trying to to find the 5' end of a gene, using Clontech's 5' RACE kit. I perform reverse transcription
on mRNA using AMV at 52 degrees for 30 min. This is performed using either a gene-specific reverse
primer, or random hexamers at a lower incubation temperature. An anchor sequence is then ligated to the
5' end of the first strand and heminested PCR is performed using nested gene-specific reverse primers
and an anchor primer. The predicted size of my PCR product should be about 400bp, but my products are
smaller (350bp approx). After cloning and sequencing, i find the extreme 5' end of my gene is missing
including the first 10-15 codons and the start codon. It is as if the reverse transcriptase has terminated
first strand synthesis prematurely (even though the template is short) and the anchor has ligated to the
truncated 5'end resulting in smaller than expected PCR products. Has anyone encountered this problem?
Can anyone help?
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