Tet selection problems in pBR322

John Nash num208jn at MBDS.NRC.CA
Fri Jan 10 17:31:09 EST 1992


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 


Try "expressing" your cells in a nutrient broth (like LB or TY, etc)
for more than one hour after transformation.  I believe Tet needs mor
than one hour to establish itself etc... I usuallly use 2 - 2.5 hr
-and pray for no sibs ;-)   Oxytetracycline HCl instead of
tetracycline HCl is also supposed to help too - can anybody else
confirm/deny this?

John.
John.


John Nash.   Nash at biologysx.lan.nrc.ca (preferred) or num208jn at mbds.nrc.ca
Institute for Biological Sciences, National Research Council of Canada,
Ottawa,  Canada.	
==>  Disclaimer:  All opinions are mine, not NRC's!  <==



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