cDNA cloning in prokaryotes

John Nash num208jn at MBDS.NRC.CA
Sun Jul 28 17:32:36 EST 1991


In article <9107262136.AA27580 at genbank.bio.net>, JMILLER%VXBIO.SPAN at STAR.STANFORD.EDU writes:
>
>  A c-DNA cloning question:
>  Our lab is planning to construct a c-DNA library for the extremely
>thermophylic eubacteria Thermotoga. Since we are new to this, we would
>appreciate suggestions/comments on the following:
>  1. The best way to make the cDNA (random primers/terminal transferrase)
>  2. Cloning vectors. We plan to use them primarily for sequencing rather
>     than expression or mapping.
>  3. Ways to avoid re-cloning highly expressed sequences.
>
>Any help on these topics would be appreciated.
>
>-Peter Markiewicz


May I ask a naive question?  Why a cDNA library and not a genomic one?

I would have thought that with the typically small size of the genomes
of eubacteria, that making a straight genomic library and screening a
slightly larger number of clones would be less hassle than extracting
mRNA from such a beast, and screening a slightly smaller number of
clones.  Working with mRNA from prokaryotes (in my limited experience)
is more hassle than it's worth - with their short half lives and the
voracity of RNAses floating around.

With a (bacterial) genomic library, it's not really such a pain
screening the number of clones one needs to, in order to find your
gene. (I wrote a little computer program for IBM clone-type computers
which implements the formula (in Maniatis) which tells you how many
clones one needs to screen (given genome size and desired fragment
size) from such a library... you're welcome to try it out).

Are you after a particular gene or genes, and do you have decent
probes?

'til later...

cheers, 
John Nash,                            Internet:  num208jn at mbds.nrc.ca 
Institute for Biological Sciences,
National Research Council of Canada,  Ottawa,  Canada  K1A 0R6.	
Disclaimer:  All opinions are mine, not NRC's!



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