Tet selection problems in pBR322

ww40 William_D_WARREN at UMAIL.UMD.EDU
Fri Jan 10 19:28:00 EST 1992


>In article <9201101859.AA20315 at umailsrv0.UMD.EDU>,  I wrote:

...stuff deleted 
>>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 !
...more stuff deleted

 then John Nash wrote:
>
>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?

Yes we tried this - even after 2 hours "expression" without
antibiotic the effect was still there (ie the ratio of Tet resistant
colonies to Amp resistance colonies remained 1:10) - and for our
purposes sib production must be avoided at all costs.

Can anyone help with info on the Oxytetracycline ????

Yes, using a different cell line or "inducing" the tet gene
could well help (thanks for the suggestions Michael Benedik 8-)

BILL
Center for Ag Biotech
Univ of Maryland




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