HELP! Lane background in Northern's
kgilbert at cmp.hmc.psu.edu
Tue Nov 22 14:42:41 EST 1994
I've been having a problem with very high non-specific binding
restricted to the entire length of all lanes on a northern blot. Here
are the specifics:
1.RNA obtained from whole lung (rat) by Tri-Reagent protocol. (Molec.
2. Samples were run through Molec. Res. Center oligo-dT columns
3. 5 ug of poly-a+ enriched RNA fractionated on agarose/formaldehyde
gels and transferred to nylon membranes.
4. Rat cDNA probe labeled with random priming. Probe is ~1.9 kb
5. Prehyb/Hyb soln is modification of Church and Gilbert (PNAS
81:1991-95, 1984); Final concentrations of components are 1% non-fat
dry milk, 1mM EDTA, 7% SDS, 0.5M Na2HPO4. Hybridize at 65 degrees C.
I get a nice band where it's supposed to be, but also get high
non-specific binding in the entire lane as well. It's not nearly as
bad with some 40-mer probes I'm using; just the cDNA's.
Also, I can still see ribsomal RNA ghosts in these poly-a+ samples.
Has anyone else had these problems? Please comment in this newsgroup
or by e-mail.
KA Gilbert, Ph.D.
Dept. Cell and Mol Physiol
Penn State Univ, Col of Med
Hershey, PA 17033
e-mail: kgilbert at cmp.hmc.psu.edu
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