fresh transformations for protein expression??
Frank van de Loo
fvandelo at ca.uky.edu
Fri Jan 5 08:28:18 EST 1996
Torsten Boerchers <borcher at uni-muenster.de> wrote:
>The more we were surprised that we now on two occacions
>with similar proteins encountered problems (i.e. no
>expression) which were solved by freshly transforming
>the E.coli (BL31(DE3)) with the expression vector instead
>of starting from a plate. So fine, that works, but I would
Me too: same problem, no explanation. However, I find I can avoid
fresh transformations every time by making a a glycerol stock
immediately, and then inoculating from this. I also went to the BLR
strain (RecA-), but don't feel that this is any better.
An aside: I spent quite a bit of effort learning how badly it can
work to combine a T7lac (as opposed to plain T7) promoter and the
pLysS-containing host. This potential problem is actually
illustrated very nicely in Fig 2 on pg 25 of the Novagen cattledog.
All the best,
Frank van de Loo.
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