Smiles on Southerns

Paul N Hengen pnh at fcs260c2.ncifcrf.gov
Wed Jul 29 10:13:52 EST 1992


In article <1992Jul28.110853.24275 at gserv1.dl.ac.uk> MORRIS at ch.unige.cmu
("Mike Morris, Geneva") writes:

>Why do bands smile (not across the gel, but within each band) on 
>genomic Southerns?

I've corrected the problem in several ways:

1. I hardly ever run a Tris-acetate gel anymore. The Tris-borate gels
produce a much cleaner band (in my hands).

2. I never use glycerol in the loading buffer. Ficoll or perhaps
sucrose is better.

3. I start off the gel run with a very low current for about 5 to 10
minutes until the dye front is most of the way into the gel. This
allows the DNA to uniformly enter the gel at the side of the well.

4. I always stain with Ethidium Bromide AFTER the gel run.

-> Problems can occur when there is overloading with DNA, the
concentration of agarose is too high, or you've pushed it so
that the gel runs faster than normal. This causes a trailing
effect, mostly due to shearing of the DNA. If you're using very
large fragments of DNA for Southerns, you may have to run the
gel very slowly and keep the concentration of agarose to about
0.8 %. Also, make sure the buffer within the gel is the same as
that in the gel box. 

Good Luck. -Paul.



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