Plasmid loss after restreaking

[Graham Dellaire]

>   ddh at unlinfo.unl.edu (dinakar bhattramakki) writes:
>  Hi netters,
>  
>  I have a problem which I think was discussed in detail very recently. 
>  Could any one post the conclusion of that discussion?
>  "PROVERB IS NOT A PROVERB UNLESS YOU EXPERIENCE IT"
>  
>  My problem: I transformed Sure18 cells with recombinant pBluescript 
>  plasmid. Selected the white transformants on LB+Amp+X-gal+IPTG plates.
>  I did a master plate of half a dozen transformants. I was unable to 


Didi you restreak from the master plate...?

Often what will happen is that when you just pick a mixed colony (from your master plate...
unless you streaked your first pick and then used a single ampR colony for your master plate)
that when grown in broth will end up with a large population of bacteria without plasmid and they 
willl out grow your plasmid carrying bacteria.  End result your plasmid is lost from culture


Graham

So bottom line always restreak your first pick and then make a master plate from the restreaked 
"pure" colonies.


Graham Dellaire

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