Hi everyone,
I was hoping somebody may be able to help out with a recent (ie. MONTHS!)
problem...with Northerns. Several of us in the lab are currently
attempting to get those gorgeous Northerns everybody dreams about
(literally!) but we have encountered various problems of different nature...
Following a standard Sambrook/Maniatis protocol, we often find that our
probe will wash off immediately in the first wash (1x SSC/1%SDS, RT). We
are using 3 different probes and therefore rule out a specific "probe" problem.
Using actin probe as a control often works well but will give quite a
bit of non-specific binding to rRNA....we can also get this non-specific
binding with our other probes (a blocking agent problem you say....we
have tried new Denhardts, various different herring sperm DNA
samples...to no avail...).
All of us have done Northerns successfully before using the same
protocols...very little non-specific binding etc...others in our lab
also, but we seem unable to solve this problem. The answer continues to
elude us. We are now considering making polyA RNA to avoid the
non-specific binding problem, but as I am sure you can understand...it is
very frustrating when you have had a seemingly routine protocol working
before...then suddenly it doesn't...for more than one person. Is anyone
else experiencing a "Northern problem" - could it be membrane-related?
We have tried fixing the RNA through both baking and UV-crosslinking.
Same problems. Arrgh! Any advice would be MOST appreciated! THANKS!
Maggie
Marguerite Evans
School of Biochemistry and Molecular Genetics
UNSW Sydney 2052
NSW
Australia
voice: (+61)(2)385 2030
fax: (+61)(2)313 6271
email: p2158740 at acsusun.acsu.unsw.edu.au
