cDNA synthesis > 6kb
Mr. S.J. Ennion
sennion at hgmp.mrc.ac.uk
Tue Oct 3 12:12:39 EST 1995
Please can anyone give me some tips on making a cDNA library with large inserts.
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.
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