benedik at uh.edu
Tue Mar 2 13:28:55 EST 1999
In article <7bh8st$ev1$1 at mserv2.dl.ac.uk>
"Malay" <curiouser at ccmb.ap.nic.in> writes:
> -----Original Message-----
> From: Malay <curiouser at ccmb.ap.nic.in>
> To: bionet.molbio.methds-reagnts mail newsgroup <bionet-news at dl.ac.uk>
> Date: Monday, February 22, 1999 10:42 PM
> Subject: Complementation
> >Dear Netters:
> >I am trying to complement the strain UQ285 of E.coli, carrying a
> >sensitive mutation of sigma-70 gene with a genomic library of a Pseudomonas
> >strain. I transform and plated several dilutions of the transformed cells.
> >When I plate around 400-500 cells per plate I get nothing. But when I plate
> >almost a lawn of cells I get some colonies growing but very poorly.
> >My question is as follows:
> >Does the density of the plating has anything to do with the complemation of
> >a temperature sensitive strain? If so then what is the ideal plating titre?
> >Any comments or suggestion will be appreciated.
> >Malay K Basu
> >curiouser at ccmb.ap.nic.in
Its hard to say what is happening. When you are plating at such a high
density you may be finding complementing plasmids that are present
rarely in the library, or you may be selecting for revertants or
pseudorevertants of your host strain. Why don't you streak these small
colonies out again at high temperature to purify them, then isolate the
plasmid and see if it complements with high efficiency or not.
Department of Biology and Biochemistry
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