cells BRL(DE3)pLysS

3Dom 3dom at caramail.com
Wed Mar 18 06:12:21 EST 1998


In article (Dans l'article) <6e0nr3$dha$1 at nnrp1.dejanews.com>,
ruben.varela-calvino at kcl.ac.uk wrote (écrivait) :

> Hi everybody:
> 
> I'm using the pET vector for cloning and expressing my proteins. The problems
> come when I try to transform the BRL(DE3)pLysS cells. I follow the Novagen
> protocol (and cells) but I only get 4-5 colonies and not in the middle of the
> plate (I use LB plates with Amp, and then spread Chloramphenicol and
> Tetracycline on then, leave dry before plate the transformation).
> Any suggestion to improve the trasnformation?
> Thanks in advance.
> Ruben Varela-Calvino
> 
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after electroporation, put immediately your bacteria on your plate,
becasue DE3pLysS cells are very hard to manipulate ; all the time you a
lysis.
i use 1500 volt on E.coli pulse system ( BioRAD ), perhaps use less Amp .

dom (bruneau at versailles.inra.fr)



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