SS Nytran-- Please Help!

Timothy Bushnell bushnt at rpi.edu
Tue Nov 22 01:53:51 EST 1994


In Article <3aqigv$eq2 at post.its.mcw.edu>, dmeier at post.its.mcw.edu (Daniel
Meier) wrote:
>Dear All,
>
>  Over the past years I have successfully used Schleicher & Schuell Nytran.
>About a month ago, our supplies ran out and we purchased additional SS
>Nytran.  Since our previous purchase, Scleicher & Schuell changed the
>formulation of their Nytran (apparently due to patent reasons).  When we use
>this new Nytran with our old hybridization conditions we have significantly
>high backgrounds (very dark at 3 days exposure).
>  We hybridize at 42 C with 50% formamide, 25 mM KOP4 (pH 7.4), 5x
>Denhardt's, 50 um/ml herring sperm DNA, and 5x SSC.  We wash blots 2x at RT
>for 15 min each using 2X SSC,0.1% SDS and 2x 15 min each using 0.25X SSC,
>0.1% SDS at 57C.  Our probe was gel-purified from a 1.5% Boehringer-Mannheim
>agarose gel.  Any suggestions how we might lower our background using these
>membranes?  Can you suggest a membrane that works well for you and allows
>multiple stripping and reprobing?
>
>Thanks much,
>
>Dan Meier
>dmeier at post.its.mcw.edu

Hi Dan,

    A couple of comments.  I have noticed similar problems with the "new"
Nytrans from S&S.  I had little success with their tech service, for the
membrane is supposed to be compatable with old protocols.

    One change that we found that worked is to add from 0.1 to 1% SDS to
your prehybe buffer.  This seemed to cut down on background significantly.

    Alternativly, you can try the BioRad "Zetaprobe" membranes, which work
well for us: low background, easy to use etc, etc.  If you use these, I
would follow their protocol exactly.

    Hope that this helps.
Timothy Bushnell
Plant research group

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