Alkaline vs. High-salt transfer of Southerns

Anita Gould anita at accord.cco.caltech.edu
Sun Jan 29 23:35:35 EST 1995


In article <stauth-250195110819 at darenmac.usgmrl.ksu.edu> stauth at crunch.usgmrl.ksu.edu (Darren M. Stauth) writes:

   Just wondering if anyone had any experience with transfering DNA by the
   alkaline method for Southern Hybridization and how it compares to
   high-salt(SSC) transfer.  Through various lab manuals I've learned that
   alkaline transfer onto charged nylon takes a shorter period of time(2hrs
   vs. overnight), binds DNA better(no need for UV crosslink), has higher
   background.  It seems superior to SSC method.  Any comments appreciated. 

Yep, crosslinking with alkaline transfer on charged nylon is
superfluous, & will actually decrease your signal.  The background
definitely is higher, so if you have any problems with that you may
want to go back to SSC (depending on what kind of blocking you're
using, & how well it deigns to work for you -- I'm having problems
right now, & it seems to depend on the phase of the moon).  Aside from
that, yes, alkaline transfer is great.  But you can also get 2-hour
transfer using SSC by doing a downward transfer instead of upward.
One warning on alkaline downward transfer: if you use the Schleicher &
Schuell Turboblotter, *don't* use the very-high-salt (3M, I think)
buffer they recommend -- it will flush the DNA right thru the membrane
(even with 0.2um nylon.  That's with neutral nylon -- never tried it
with charged.)  Use the Maniatis alkaline transfer buffers instead.


--

                                   -Anita Gould
                                    anita at cco.caltech.edu



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