Ferret Brain cDNA Library Mystery

Michael Morales mmorales at acpub.duke.edu
Tue May 2 11:54:40 EST 1995


What's so funny about ferret?  Actually, we should get together as you and I 
are probably the only "ferret molecular biologists" on the planet.  So, what 
I do know is that ferret voltage gated channel sequences are often very close 
to human channel sequences, so I'd try human oligos on your RT-PCR.  Also
give touchdown PCR some thought.

Good luck,  Mike Morales   



Jim Hutchins (hutchins at fiona.umsmed.edu) 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.

: 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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