Ferret Brain cDNA Library Mystery

Jim Hutchins hutchins at fiona.umsmed.edu
Fri Apr 28 06:56:47 EST 1995


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.

The library construction went well, as far as I could tell, and some
standard checks (e.g., % of clones carrying G3PDH) looked pretty good.
However, I have been unable to find positive clones for muscarinic 
receptors, despite using a wide variety of probes, including sequences
which are conserved with 90-95% sequence identity between (say)
Drosophila and human.  (I presume this is the sequence in the ACh 
binding site.)

Simultaneously, attempts to use the same probes as primers in RT-PCR
of rat brain mRNA (where the sequence is known, and there is 100% match)
have been a failure, too.

I expect that these are extremely rare messages, but this is ridiculous.

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.


--
Jim Hutchins
Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr
http://fiona.umsmed.edu/~hutchins/  ***  mailto://hutchins@fiona.umsmed.edu
``There are two tragedies in life: one is not getting what you want.
The other is getting it.''  Oscar Wilde, _The Importance of Being Earnest_



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