RecA-minus cells for T7 expression

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Thu Oct 26 03:41:26 EST 2000


In article <jRLJ5.70$x3.929 at uchinews>, the eminent Emir at The
University of Chicago wrote
>I would appreciate someone recommending me an E.coli RecA- strain that could
>be used for lambda CE6 induction of expression from a pET (T7)-type
>construct. BL21 (not DE3) fits the purposes of CE6 induction (see
>Stratagene's CE6 induction manual), but it is RecA+. I don't perfectly
>understand the details of what makes BL21 cells permissive for lambda
>lysogeny, and it is difficult for me to figure out which of the conventional
>RecA- strains (e.g., DH5alpha, HB101, JM109, XL-1 Blue) would allow
>infection by the phage and insertion and expression of the T7 polymerase.
>Thank you.
>Emir
>
>

Basically they should all work fine. I think CE6 is a fairly standard
infective lambda and does not depend on supF. Easy to find out. 

Grow some cells of each type in TB (LB minus yeast) O/N with 0.2%
maltose. In the morning resuspend in one half volume of 10mM MgSO4
(sterile). 

Now take a 1ul loop and streak some CE6 on the surface of four plates of
TB.

Next take 100ul of cells into 3ml of TB plus 10mM Mg 0.7% agar i.e TOP
agar at 45C and pour over streaked CE6.

Leave to set then incubate at 37C for a few hours. 

If you get plaques then the cells are OK to use when transformed with
your T7 expression vector.

If you want a good experimental book with good methods, try Experiments
with Gene Fusions from CSH. Not quite as heavy as Miller's Experiments
in Molecular Genetics, also from CSH.    

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

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






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