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.
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