ARGH! Northern Problems (again!)

Patrick HJ Falckh p.falckh at rmit.edu.au
Sun Jan 14 20:11:54 EST 1996


Robert Fisher <fisher at radonc.unc.edu> wrote:
>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
>

Rob,
   Why dry it down a second time.  Provided that your initial solubilisation is not too dilute why not add the denaturing loading buffer to a small aliquot of your original solublised RNA.  A method out of FOCUS (10 I think ?!) allowed the use of 4.5 ul of sample (either the required amount of RNA in that volume or add SDW to make the volume 4.5 ul) to which was added 15.5 ul of sample buffer which was made up as :
                 1.0 ml deionised formamide
               350.0 ul formaldehyde
               200.0 ul 10x MOPS
    - then aliquot into 300 ul units to store at -20C.
If you add 1 ul of 1.0 mg/ml ethidium bromide and THEN heat denature, then add 4.0 ul of loading buffer (6x stock).  The sample loaded can be visualised on the gel after running without further staining or other treatment and the RNA is approx. 10x as bright compared to adding the EtBr after denaturing.

This saves heaps of time and there is little chance of other difficulties affecting the result

Hope this ios helpful

Patrick

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