full length lacZ ?
jgraham at bronze.ucs.indiana.edu
Thu Dec 9 18:12:09 EST 1993
When you get that plasmid, you will have very careful as full length
lacZ on a plasmid is a very unstable situation. Although this must
be widely known, it is not widely reported. In a course we offer here
students merely grow an E.coli strain containing pSKS105 and collect
lac deletion mutants that arise at a high spontaneous frequency.
Athough a single copy lacZ on a phage or intergated into the
chromosome might be an alternative, I would seriously consider an
alternative reporter gene (eg. galactokinase, luciferase, cat, ect)
which will unfortunately be more difficult to assay. Another problem you
may see is that even with a pUC copy number of lac repressor, the
basal level of B-gal activity remains high enough to create blue
colonies on X-gal plates. An alternative like the weaker arabinose
promoter can reduce this uninduced level, though still not below
the X-gal threshold.
pSKS105 is from:
Shapira, SK, Chou, J., Richaud, F.V., and M. J. Casadaban. 1983.
Gene 25, 71-82.
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