ARGH! Northern Problems (again!)
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
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?
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