There has been a somewhat related discussion in the lab the other day.
If some colonies might express well and other not so well, why don't I
just scoop up loads and start my culture from them? This way I'll be
more certain to get the clones that express.
The clones that don't express will dilute the yield, but I doubt they'd
outcompete the rest, would they? For me, the start-with-plenty
procedure has always worked well, but some insist on starting from
single colonies.
Andreas
James Stroud wrote:
On Feb 2, 2008, at 2:15 AM, M T wrote:
One classical way to optimize expression level is to screen culture
conditions.
For my proteins, I solved my expression problems by changing the
expression vector to a pET or changing a pET 20 to a pET 30 (if the
protein is toxic).
But keep in mind that a low but folded expression is better than a
high expression to inclusion body.
Anecdotal evidence also suggests to try picking several colonies from
the original cloning procedure and doing test expressions with each.
Some clones express better than others even though they should in
principle express identically. I have no idea what the mechanism for
this differential expression could be.
--
James Stroud
UCLA-DOE Institute for Genomics and Proteomics
Box 951570
Los Angeles, CA 90095
http://www.jamesstroud.com
