S&S Rad Free system

[Klaus Salger]

Huu Minh Tran (htran at ITSA.UCSF.EDU) wrote:

: Does any one have exeperience with the S&S Rad Free System for Northern 
: Blot? I will be very appreciated if you have any suggestion.
<snip>
: There is also another problem which I don't know whether I UV crosslinked 
: too long which destroyed the RNAs or too short which would not 
: permanently bind the RNAs.  I crosslinked using Fotodyne 310 (300nm) 
: transilluminator for 1'30'', 3', 7', respectively; and baked till dry at 80 
: Celcius for 15'.  After I hyb.ed, I stripped the probes and stained the 
: blots with Methylene Blue, I don't see any RNAs retaining on the blot.  
: What was happened ?  I don't know.  One thing I forgot to mention that I 
: use the non-formamide hybridization buffer for my Northern hybridizations.
: Please, reply to my e-mail htran at itsa.ucsf.edu
: Thank you very much and looking forward to receiving replies.
: Huu Tran

I don't know this S&S system, but had the same problem when using the
DIG system. It turned out that I have an RNase contamination somewhere,
so that I didn't get a signal after the first stripping. It took some
time for me to realize that crosslinking was not the problem.
I tried to get rid of the contamination but didn't succeed (yet).
Now I include about 0.1% DMDC in the staining solution. This works
even in 100mM Tris (don't know why). Pretreatment of the Tris buffer
doesn't work.
Hope this helps
  Klaus
--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
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