Quickest method to visualize plasmid presence in bugs?

Mr. I.S. Viney iviney at hgmp.mrc.ac.uk
Tue Jan 30 04:13:00 EST 1996



On 29 Jan 1996, John Dixon wrote:

> 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.
> 
> 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
> 

Dear John,

	We used to use a very fast simple "cracking" procedure to test
presence/size of plasmid clones when I worked in your Department (on the
Downing Street site).  We used to resuspend a toothpick of cells in 200ul
modified agarose gel loading buffer, wait 15 mins, centrifuge 15 mins
(13000 rpm in microcentrifuge), then load 15-30 ul and subject to
electrophoresis.  The problem was that some bacterial strains give a very
gluey lysate which doesn't load, although our lab strain (AB1157) works
beautifully, and there were plenty of tricks developed to load difficult
strains.  Maybe if you can centrifuge your 96 well plates you can adapt
the procedure.  The only modification to the loading buffer, which was a
standard Ficoll/TAE/SDS/Bromophenol blue buffer was that the SDS
concentration was increased from .5% to 5%. 

Hope that was of interest, I'd be happy to provide more details. 

Ian Viney
Imperial College
LONDON





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