Southern blots

George Rutherford gruther at bilbo.bio.purdue.edu
Tue Nov 30 15:02:01 EST 1999


In article <82155q$qnv$1 at nnrp1.deja.com>, vivanova at my-deja.com wrote:

> HI, all!
> 
> Here I have a question to all the Southern experts:
> 
> What's the best way to do a Southern transfer? Maniatis uses a HCl
> followed by 0.5 N NaOH to depurinate and nick the genomic DNA, then
> transfers in SSC. Has anyone of you got a protocol that uses NaOH as the
> transfer buffer? Any useful suggestions and comments would be greatly
> appreciated!Previous experience has given somewhat fuzzy bands om the
> film. Could that be improved in some way? Is the HCl/NaOH contributing
> to the fuzziness?


Hiya,

Maniatis covers alk transfer somewhere in Chapter 9, I believe (latest
edition, not that it's that late). Works for me. You have to use nylon,
though. NC will fall apart. It's unlikely that HCl/NaOH is the cause of
your fuzziness; it's more likely due to electrophoresis conditions/buffers
or DNA isolation methods (you may have some mild degradation). Good luck.

George




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