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My Northerns won't work!

Patrick HJ Falckh p.falckh at rmit.edu.au
Wed Dec 20 17:49:31 EST 1995


Sprankle at ciit.org (Cathy Sprankle) wrote:
>After 5+ years of running Northerns pretty much without any trouble, a few
>months ago they stopped working for me.  I am getting a hybridization
>signal on my blots, just not much of one (maybe 25% of what I should be
>seeing).  RNA appears to be intact (the signals I'm getting are bands, not
>smears), and hybridization with a control probe confirmed that the problem
>is with the blots themselves rather than the hybridization conditions or
>procedure or the probes I've been using.  I compared UV crosslinked blots
>to baked blots, no difference.  Also compared two different packs of
>Hybond N, again no difference.  Where this leaves me is:
>
>--something is chewing up my RNA during the gel washes, during the
>transfer, or after the transfer, or
>--something is wrong with my transfer (old-fashioned upward capillary blot
>procedure).
>
>I would appreciate suggestions about where I should go from here.  Thanks
>very much.
>
>Cathy
>
>-- 
>Catherine Sprankle
>CIIT
>e-mail:  sprankle at ciit.org
>Opinions expressed (such as they are!) are strictly mine and do
>not reflect those of CIIT.

Hi Cathy,

I now you've probably thought of it but it could be that your 
reagents/transfer buffers are not sterile or devoid of RNases.  I had similar 
problems with RT/PCR reactions until I worked on the most obvious problem ... 
the autoclave was malfunctioning and even though it passed a maintenance check 
I had to increase the sterilization time by 2 fold !!  It solved my problem, 
it may solve yours.
The other question is whether other people use your buffers and thereby 
introduce RNases; hopefully inadvertently.

Regards

Patrick



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