smori at nmsu.edu
Fri Jan 13 00:09:14 EST 1995
St George's Hosp Medical School (fenechc at sghms.ac.uk) wrote:
: 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?
What MIGHT work is using superscript II. It may be that the AMV is
dropping off before it reaches the end of the message. Also superscript lacks
the Rnase part that degrades the RNA. You could also ( in combination with
suprescript) increase the time of cDNA synthesis from 30 to 60 minutes.
Shahram Mori _/\_
Program in Molecular Biology _\ /_
Dept. of chemistry and Biochemistry Box 3C \_ _/
NMSU Las Cruces NM ||
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