Tet selection problems in pBR322

William J. Buikema wjb1 at quads.uchicago.edu
Mon Jan 13 15:11:37 EST 1992


The tetracycline resistance determinant in pBR322 does not require induction,
which is not the case with other tet determinants, such as from Tn10, which 
also contain a repressor gene that must be bound to tetracycline in order to 
be inactivated.  pBR322's tet gene is constitutively on.
	I believe that in general it is the case that tet selection is a
'stronger' selection than Amp, and apparent transformation efficiencies fall.
In my hands, tet selection at 12 ug/ml is rather high.  I have used 5 ug/ml
successfully with many strains of E. coli.  One technique you can try is to 
overlayer a tet plate with a thin layer of plain LB-agar or LB-agar with a 
low amount of tet (say 2.5-5 ug/ml), and see if your efficiencies improve.

Good luck,


William J. Buikema              internet: wjb1 at midway.uchicago.edu
University of Chicago             bitnet: wjb1%midway.uchicago.edu at uchimvs1
Dept. of Molecular Genetics &       or    wjb1 at midway.uchicago.edu 
    Cell Biology, CLSC 313
920 E. 58th St.
Chicago, IL  60637



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