Sub cloning

[Vladimir Svetlov]

In article <5rso9l$98g_001 at leeds.ac.uk>, BMBLDO at leeds.ac.uk (L.D. Ofner) wrote:


Your control with vector - did you ligate it or just transformed the cells
with the prep? It might be cut but not dephosphorylated. I routinely use
Shrimp phosphatase right in the overnight digest -it's compatible with most
restriction buffers (as salty as H by Boehringer).
The idea about possible toxicity is interesting - does your expression
vector leak at non-inducing conditions? 
one thing that surprises me is that you get "loads" of colonies regardless
of the insert/vector ratio. This increases the probability that your vector
is not dephosphorylated properly.
Good luck,
V.