Problem Blotting of Pulsed Field Gels

Rafael Maldonado rafael at howard.genetics.utah.edu
Fri Jan 10 14:51:15 EST 1997


On Thu, 9 Jan 1997, Mike Chao wrote:
> >   Depurination in 0.25M HCL for 0,10, or 20 minutes
> >   Neutral vs. alkaline blotting
> >   Traditional capillary blotting (24 hrs.) vs. vacuum blotting
> >   1% 'standard' agarose vs. 1% Fast Lane agarose

You may use UV iradiation of the gel instaed of the depurination. It is 
possible that the depurination it is not working for some reason. 3-5 
minutes of iradiaton will be enough.

Do you know in which step you have the problem? it could be a problem of the 
filter. I would not use a N+ membrane for this, a regular Hybond worked 
best for me. Is there too much DNA remaining in the gel after the 
transfer? O/N transfer by the traditional method seemed also to work 
better in my hands.


Rafa
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