ARGH! Northern Problems (again!)

Robert Fisher fisher at radonc.unc.edu
Fri Jan 12 14:14:22 EST 1996


After months of successful northern blots, i have run into a problem.  When
i run my total RNA out on a 1.2% agarose/formaldehyde gel, the 18S and 28S
bands
vary in intensity from one sample to another, although according to the spect.
a260 readings i am loading the same amount of RNA.  There doesn't appear to be
much degredation.  The RNA is isolated by the GITC/phenol method.

This may seem silly, but could this be due to a solubility problem?  I don't
overdry my RNA pellets from the EtOH ppt, and I resuspend them in DEPC h20.  I
usually let them dissolve and leave them at 4 degrees c for an hour or so
before i freeze them down.  I measure out the proper amount of RNA solution
(according to our spect.) to give me 10ug, then dry it down in the speedvac.
Again, i am careful not to overdry.  i resuspend in denaturing loading buffer
and heat at 95 degrees for 5 minutes to denature prior to loading my gel.

can anyone see anything obviously wrong?

-rob fisher




More information about the Methods mailing list