ss cDNA Sequencing

From: Andrea Erica Varga (andrea.varga@student.adelaide.edu.au)
Date: Thu Mar 29 2001 - 21:48:22 EST


Hello.

I have some human ss cDNA from primer extension and was wondering what the best
method was to sequence it.

There do not appear to be any vectors which it can be inserted into, as they
all require dsDNA.

That leaves me to sequence it manually with Sequenase v2.0 or equivalent. My
main questions are:

What should I use as a template?
Should I do an RT using oligo(dT) primer, or should I use my specific primer
(from the p. extn) for the template? My main concern is the orientation of
the strand. My 5'-3' mRNAs and my 5'-3' primer in the same orientation. Hmmm.

Please help.
Andrea.
-----------------------------------------------

Andrea Varga

       The Department of Surgery
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