I am sorry. I forgot to specify what kind of wet blotting method I was lookig for in my previous message.
I have been looking for a protocol for DNA wet ELECTROBLOTTING onto Hybond N+ membrane. Can anyone tell me one?
Is it O.K if I depurinate the DNA (I have large, > 10 kb fragments) in 0.25 M HCl, then denature the DNA by agaitating the gel in 0.4M NaOH, then equlibrate the gel by rinsing it in 0.5XTBE several times, then do the electroblott in 0.5X TBE? Is this tank buffer good? Does not the DNA renaturate? Will the DNA bind the the Nplus membrane in single starnded form in this buffer (pH is ca. 8.3)? Baking or NaOH fixation is better?
I would appreciate any advice and comments.
(Edited by jbak at 7:07 pm on May 24, 2001)