undenatured Southern blot usable?

Kirov kirov at medfac.acad.bg
Sun Nov 9 07:32:48 EST 1997


Hi,
What's going on with these Southerns? Everybody is forgeting something!
However this mistake compared to the wrong SSC is much more unpleasant.
Though there aren't too many chances I guess you can try to save your
blot by NaOH denaturation and immediate immobilization. But if you have
already used UV to crosslink the DNA onto the Hybond, now you will have
to use baking. Two much UV irradiation will cause much less signal. In
any case you will have weaker signal than usual because of the mistake.
Since I am only speculating I should warn you that it is possible that
you shan't have detectable signal at all (depends on the DNA you are
blotting and and so on). If it is not something very important - do it
again.
There is one more possible approach if your DNA is >1kb: use HCl and
then NaOH and immobilize almost as you do it with the gel. Then probably
some of the DNA will be hydrolyzed, some fragments will be denatured and
will be accessible target for probe hybrizization.
All of these may not work.
Good luck!
-- 
Steven Kirov
PhD student
Lab. of Molecular Biology and Genetics
Dept. Of Biochemistry
Medical Faculty
Sofia
Bulgaria



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