Blue/white colony selection problem.

[Duncan Clark]

In article: <34pe3i$qg9 at nuscc.nus.sg>  medp3056 at leonis.nus.sg (G Selvarani) writes:
> 
> Wises Namwat - SCMI - 3636183 (gscwnw at MUCC.MAHIDOL.AC.TH) wrote:
> : Hello  netter,
> :   	I have some problems in transformation technique. pGEM7(+/-) was 
> : used as a vector for the DNA insert, this ligated product was then 
> : transfect into competent E. coli(DH5alpha). Blue/white colonies 
> : growing on amp&x-gal plate can be use for selection the success insertion 
> : clone, the white colonies. Theoritically, the white colonies must be 
> : bigger than the blue ones. But the smaller white colonies always appear 
> : around the blue colony. These smaller clones contain the different size 
> : of plasmid. Does anybody know what are these smaller colonies, and where 
> : does their plasmid come from?
> : 	Thanks in advance.
> : ----------------------------------------------------------
> : Wises namwat             email: gscwnw at mucc.mahidol.ac.th
> : Dep. Microbiology,       Tel.66-02-2461358-74 Ext.4611    
> : Fac. Science,
> : Mahidol University,
> : Bangkok, Thailand.
> : ----------------------------------------------------------
> :   
> Hi! 
> We've encounted such things. In fact, if we spread our plates too early 
> in the day the sattelite colonies(the little one around the big colony) do 
> appear.Apparently the satellite colonies (competent colony)grows 
> surrounding the real colonies ones the ampicillin runs out around the area
> they do not contain any insert or plasmid. dna appears with extractions 
> maybe due to contamination of chromosomal dna.
> that,s all.
> 

Try using 20mg/ml ampicillin + 80mg/ml methicillin (Sigma Cat. No. 
M6535 approx US$90/25g). You will cut out the satellites. I would not 
advise using this for liquid cultures with IPTG induction. I found 
that positive expression clones lysed overnight. Good way of telling 
that I had the correct construct but not the best way to produce the 
enzyme concerned!  

Good luck

Duncan

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Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
G4ELJ                               | Compuserve:  100015.1406 at compuserve.com
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