fresh transformations for protein expression??

[Frank van de Loo]

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.