Cloning problem

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Feb 9 05:06:58 EST 1999


In article <79omnt$dr0$1 at mserv2.dl.ac.uk>, Malay
<curiouser at ccmb.ap.nic.in> writes
>Hello Netters:
>
>I am trying to clone RpoS (stationary phase sigma factor) gene from a
>Pseudomonas strain. I used heterologous probe from P. aeriginosa, E. coli
>and also used a oligomer probe. In all these cases it shows a distinct band
>on gel about 3.5 Kb size. I cut out the band and ligated to pUC19/pBluscript
>vector, transformed DH5alpha cells and screened with the same probe. But in
>none of these cases could pick up any positive clone. Thinking that, being a
>metabolically crucial gene it might create some problem, I did a digestion
>to cut the original band into half and again tried to clone two fragments
>separately using the same strategy. But it again failed.
>
>Could anybody tell a fast and alternative strategy to clone this gene. Any
>suggestion will be appreciated.
>
>Thanking you,
>
>Malay
>curiouser at ccmb.ap.nic.in
>

It is most likely lethal. So clone opposite to lac. Use a well switched
off promoter/operator/inhibtor expression system, maybe arabinose? Use
a lower copy vector. Use lower growth temperature. You basically need to
reduce the basal level of expression to zero. Have a look in the bionet
archives, as this topic occurs often and other hints will be found.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk



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