what conc antibiotics for Carb/Kan/Tet cocktail?

John Dixon jpcd0 at mole.bio.cam.ac.uk
Thu Aug 22 08:20:41 EST 1996


Hi, a simple microbiology question I hope:-

If you want to select for bacteria which have multiple antibioic
resistance, do you use the same concentration of antibiotic as you would
if there was only one antibiotic resistance gene, or do you drop the
concentration of them to avoid hitting the bugs too hard?

Long-ish Detail follows:-

I use the DH10B:p3 (Gibco) for cloning inserts that contain supF. The p3
episome already present in this strain confers Kanamycin resistance, and
carries amber mutated genes for Ampicillin and Tetracyclin resistance.

The bugs are therefore KanR, TetS and CarbS, initially. If a supF
containing plasmid is transformed in, the supF suppresses the amber
mutations and those bugs become Tet and Carb resistant.

So you can plate these out on Kan/Carb/Tet plates and only the insert
containing bugs can grow.

Unfortunately, amber revertants spontaneously arise in the population
giving background Carb and Tet resistant bugs.

The plasimds I was using have a +type ampR gene so when I want to clone in
a supF containing insert I can only use the Tet amber for selection. 

Invitrogen who also sell a p3 strain recommend using low Tet and Carb
(10ug/ml) to avoid enriching for revertants, presumably because bugs with
a reverted wild type gene will out grow those with a supressed mutant
gene, conferring a low level resistance.

However, I either get virtually nothing or virtually everything growing on
Tet only plates (10ug/ml), depending on how long I leave them, even when I
transform plasmids that I know contain supF.

Does any one have some tips, especially other users of this system who can
tell me what conc antibiotics I should use?

Cheers and sorry for the essay,

John

-- 
John Dixon                                        Lab 44 (1223) 334131
Wellcome/CRC Institute                            Fax 44 (1223) 334134
Department of Genetics
Cambridge University    
United Kingdom                           e-m: jpcd0 at mole.bio.cam.ac.uk



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