Quickest method to visualize plasmid presence in bugs?

Dr. Duncan Clark duncan at genesys.demon.co.uk
Tue Jan 30 06:26:52 EST 1996


In article <jpcd0-2901961926050001 at macr1-2.welc.cam.ac.uk>, John Dixon
<jpcd0 at mole.bio.cam.ac.uk> writes

>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.

Hi John,

I saw a protocol may moons ago as a single sheet paper at the back of
NAR. Basically grow on rich plates and scarpe the colony off, do a small
scale alkaline lysis ie 20ul total, mircofuge and load a gel. It does
work but runs in slowly due to ionic strength. 

Alternatively as you have them gridded out you reduce the number of PCRs
required to screen by pooling aliquots of colonies in individual rows
and columns ie pool a1,a2,a3,a4,a5,a6,a7,a8 then b1 etc Repeat for rows.
For a 40 x 40 grid you can screen 1600 colonies with just 80 PCRs  

  A B C D E F G H  
1
2
3
4
5
6
7
8
8

Finally is it possible to tag your insert with a 25mer lac operator
sequence? In a lac+ E.coli ie HB101 or K802 it will titrate out all the
lacI and switch on lacZ giving blue colonies on Xgal plates.

Duncan 


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