Quickest method to visualize plasmid presence in bugs?

Rolf Marteijn Rolf.Marteijn at 94.student.wau.nl
Mon Jan 29 19:05:11 EST 1996








Hi John,

Well, a fast way of checking for the presence of plasmids (and some
estimation of the size of the plasmids) is the Cracking protocol. I
found it in a Promega Manual. You put a colony in 50 ul of 10mM EDTA,
add 2x Crack (SDS, NaOH, Sucrose), vortex, put for some minutes at
70C, cool down, add 1.5 ul 4M KCl  and 1 ul of .4% bromephenolblue,
vortex, spin for 3 minutes and put on a gel. All can be done in only
one eppje. This protocol works quite convenient, but you should keep
in mind that you get circular plasmids and some genomic DNA on the
gel. 

Also you could -of course- do a mini-prep but a quicky one. A lot of
protocols excist for this one...


Greetings,

Rolf






jpcd0 at mole.bio.cam.ac.uk (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.

>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

-------------
Rolf Marteijn
Wageningen Agricultural University
The Netherlands
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