cDNA synthesis > 6kb

Lauryl Maiya J Nutter lmjnutte at acs5.acs.ucalgary.ca
Tue Oct 3 20:07:50 EST 1995


Mr. S.J. Ennion (sennion at hgmp.mrc.ac.uk) wrote:

: The mRNA(s) I want to isolate at the cDNA level are 6Kb in length.
: I have previously had problems in making cDNA to this length.
: I've tried M-MlV RT from stratagene and AMV RT at higher temperatures with
: and without actinomycin D. Each time I get cDNA but never full length 
: (I check the length by RT-PCR with primers from both the 3'end (works) and
: 5'end (doesn't work)of the mRNA). I have primed the cDNA with either oligo dT
: or a specific primer from the 3'UTR of the gene.

: If anyone can pass on their experience (with or without a kit) of making full
: length large cDNAs for library production, it would be a great help.

I have successfully made cDNA from total Drosophila RNA at least
4 kb in length using GIBCO/BRL Superscript II RT.  This is cloned
MMLV RT with three point mutations which reduces the RNAse H
activity inherent to MMLV RT to below detectable levels
(according to GIBCO).  I also recently attended a seminar put on
by Larry Mertz of GIBCO and he showed data for the reverse
transcription of complete messages up to 7.5 kb.  You may want to
give them a call and check it out.

Note that I don't own any shares in GIBCO, etc.
I'm just a grad student trying to figure out a very
complex transcription pattern using RT-PCR since library
screening didn't work.

Good luck,

Lauryl Nutter
Biological Sciences
University of Calgary
lmjnutte at acs.ucalgary.ca



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