Northerns...AGAIN!

Joy Ann Buckle g06jab at morgan.ucs.mun.ca
Mon Oct 6 08:04:35 EST 1997


Can any one give me a cluse as to what to do?  I started working with RNA
and Northern blots last September.   When I finally went to do a Northern,
it worked.  However, that was the first and only time.  The lab tech and I
have been struggling with it ever since.  For the longest time we had a
problem with black blots which turned out to be a problem with the
membrane we were using.  We have since swithced to HYBOND and while the
control probe for the amount of RNA loaded works fine, the other probe I
am using isn't working.

My supervisor and the lab tech have been using this probe for a long time.
My supervisor did all her thesis work with this.  However, the problem now
is that we make the probe and hybridize.  The counts  seem to be quite
high when the counter is placed next to it after hybrizidation.  The
problem is that the wash solution cannt go near the membrane and all the
probe literally disappears.  My supervisor also told me this past week
that I have non-specific binding.  The probe is still the same as my
supervisor's but even with the same wash solution, the probe is not
working as it should be.  It isn't working at all!

The wash solution used was:

1X SSC,1%SDS 15 min(RT)
0.5 X SSC,0.5%SDS 15 min(RT)
0.1 XSSC,0.15 % SDS 2 X15 min (RT)
0.1 X SSC 0.15%SDS 30 min @RT

We can't get past the first 15 min wash now and the probe is literally
gone.

As for the non-specific binding, I have no idea what to be doing for that
either.

Any suggestions? I am getting desparate.  I have been here a year and am
now well into my second.  My masters is only a two year project.  Please,
any info would be greatly appreciated.

Thanks,
Joy :)




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