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.
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
Now take a 1ul loop and streak some CE6 on the surface of four plates of
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.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
More information about the Methods