Problem Blotting of Pulsed Field Gels
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.
Rafael Maldonado | 'No te creas todo
room 6160 Eccles Institute of Human Genetics | lo que leas en
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