Tet selection problems in pBR322

ww40 William_D_WARREN at UMAIL.UMD.EDU
Fri Jan 10 13:50:00 EST 1992


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 




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