PFGE blotting problems

James S. Sutcliffe jamess at mbcr.bcm.tmc.edu
Tue Feb 8 11:44:48 EST 1994


I missed the original post on this issue, so I'm not sure what the problem
was. But, having done lots of PFGE blotting experiments, I'm aware there are
a few tricks to good PFGE Southerns.

1. I've seen several protocols citing the virtues of UV nicking the DNA in
the gel. This never worked well for me. Generally, 20-30 min depurination in
0.25 N HCl (as opposed to the std. 10 min. for conventional gel) is required.
Preferably with some agitation (platform shaker). Some people even change the
solution midway thru this step. 

2. Everything else is the same, except blotting time. Blotting for 2 days
seems to help significantly in the degree of transfer. This may be very time
consuming, but a lousy PFG filter doesn't do any good either, especially
considering the amount of time it takes to run some gels.

If you're having problems with the blotting, it's not a bad idea to stain and
destain the gel (again) after transfer to see if all the DNA has transferred.
The EtBr dissociates from the DNA during transfer, so restaining is necessary.

Hope this helps.

Jim Sutcliffe



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