Quickest method to visualize plasmid presence in bugs?

John Dixon jpcd0 at mole.bio.cam.ac.uk
Mon Jan 29 14:25:15 EST 1996


Hi All, 

Can anyone recommend a fast way to differentiate between bacterial
cultures/colonies that do or dont contain plasmids? Some sort of
boiling/alkaline lysis and immediate running perhaps.

More info on reason for request:- ie why I cant just plate on carb plates.

I am trying to clone the genomic flanking region of a transgene that
contains the supF amber suppressor. To do this I am trying to construct
sub-genomic libraries in bluescript which I then transform into DH10Bp3
cells. This host contains the very low copy number p3 episome; conferring
KanR and amber AmpR, amber TetR. When my ligations are plated on Carb
plates I see some blue vector background and some whites giving a rough
idea of the complexity of the library. When plated on carb, kan, tet
plates I get only a few whites, but so far these have turned out to be
amber revertants , ie empty hosts where the p3 has reverted the mutations
in Tet and Amp.

I have been tinkering with my ligation conditions and now have several
hundred of these white colonies over 20 or so plates. I tried direct pcr
to look for sequences internal to the transgene but got no +ves. I worry
that the amount of bugs I sraped into the 96 well plate was excessive for
a 50ul pcr (I should have added a clump of bugs to one of the plasmid
spiked +ve controls).

I gridded out these colonies onto another CKT plate which I could blot and
probe, but I would prefer to pick and culture O/N and then lyse in 96 well
plates and just run the lysed bugs out.

Is there an easy - ie minimal hands on time - method for this simple test?

Thanks

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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