Ferret Brain cDNA Library Mystery

Ben Stanger stanger at rascal.med.harvard.edu
Sat Apr 29 17:22:04 EST 1995

hutchins at fiona.umsmed.edu (Jim Hutchins) wrote:
> I have hit the wall with a baffling problem.  Several months ago, I 
> constructed a ferret brain cDNA library in order to sequence the brain
> muscarinic receptor subtypes.  The idea here was to get the sequence,
> so as to be able to design primers for RT-PCR.

> Are there any signs (besides the obvious ones above :-) ) that sequences
> are unclonable and unamplify-able?  I have heard of such things but would
> rather prove to myself that I am making some systematic error than give
> up on this project.  Further, I need to know whether to invest more time
> and effort on other aspects of this library, or to trash it and start
> over.
> Any general advice on "library testing" or specific advice on my predicament
> would be appreciated.

One characteristic of cDNA library construction that may vary between
species is RNA secondary structure that may prevent reverse transcriptase 
readthrough. Thus 5' sequences may be well represented in cDNAs from
one species but not another. Was your library primed with random hexamer
or oligo dT (or other), and were your probes and primers from the 5' or
3' portion of the sequence? If the library was dT-primed, you might
try using a more 3' probe and/or primers.

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