Gel purification ligation problem

Frank O. Fackelmayer Frank at Fackelmayer.de
Fri Jul 20 06:20:34 EST 2001



Chad Rappleye wrote:
> 
> From this thread it seems that everyone is pointing to UV-damage as the
> most probable culprit for failed ligations.  To avoid UV altogether, I've
> been using crystal violet to stain my gels as suggested in
> 
> "Crystal violet can be used to visualize DNA bands during gel
> electrophoresis and to improve cloning efficiency", K.N. Rand, Technical
> Tips Online 1996.
> 
> Using crytal violet, there is no UV at all.  You can sit and stare at
> the gel all you want and trim it as small as you want.  The only
> drawback is that it isn't as sensitive as Ethidium staining
> but for cloning you should be using enough DNA to see it by crystal
> violet anyway.


well, crystal violet works for visualizing DNA in gels under normal
light conditions, and MAY be good to avoid UV damage of DNA fragments to
be cloned. 

However: 
* It stains everything, including the gel tanks, plasticware, etc. Not
very practical in many labs. And it makes ugly stains on clothes...
* It ISN´T a safe alternative to EtBr, it is toxic. The MSDS of both
compounds look very much alike, and there are hints that at least
breathing the dust may cause cancer (solution listed as
non-carcinogenic, just like EtBr!). The problem is that you have to use
WAY more crystal violet to see the DNA. This is certainly not good for a
compound with ill-defined potential to be harmful.

We tried it, found it "NICE", and have returned to EtBr.

Frank




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