S&S Rad Free system

Huu Minh Tran htran at ITSA.UCSF.EDU
Mon Nov 14 16:31:22 EST 1994


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.

The problems that I am now currently dealing with are patched background 
with faint signals.  I asked the technical services at S&S and even sent 
them the outline of the procedure which I followed.  The replies were 
that my procedure seem okay and should give good adequate results.  Yet, 
I haven't had any good Northern blot.  I used 0.2 Micron S&S Max Strength 
Nylon membrane and 1X MOPS gel with 0.41 M formaldehyde and downward 
transfer using 10X SSC buffer overnight.  RNAs were intact and good since 
they were stained with EtBr and photographed for hard copy.

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




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