Tet selection problems in pBR322

[Michael Benedik]

In article <9201101859.AA20315 at umailsrv0.UMD.EDU>, William_D_WARREN at UMAIL.UMD.EDU (ww40) writes:
>We are having some problems with selecting for Tet resistance with
>pBR322.
>
>In a control experiment we transformed unmodified pBR322
>into JM83 and observed ten-fold less colonies when plating on 12ug/ml
>Tet than when plating an identical amount on 50ug/ml Amp !
>
>This shouldn't cause any major difficulties for most cloning applications but
>we are trying to recover rare deletion events in an Amp sensitive "Tet
>resistant" construct in pBR322 and absolute numbers are really important.
>The results above indicate that we are recovering roughly one tenth of the
>deletion events than we would if we had put the insert in the Tet gene.
>
>Apart from recloning our construct into another replicon that uses some
>other selection procedure, does anyone have any suggestion as to how we can
>improve our Tet resistant plasmid recovery ?
>
>BILL
>Center for Ag. Biotech
>Univ of Maryland 

1) are you giving enough time for expression of resistance before
	plating out on selective plates?
2) during the expression period you could try inducing the tet promoter
	(it is regulated) by adding a low level of tet to the liquid
	culture during the expression phase. About 1ug/ml should be
	enough to induce without being lethal.
3) try a different e.coli strain. JM83 may just not be happy on tet
	plates so you will get reduced plating efficiency.


	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik at UH.EDU	BITNET: Benedik at UHOU