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