RNA probes running inconsistently

greenspun.com : LUSENET : PACE Technique : One Thread

Iíve had challenging experiences with my first PACE experiments. One of the major problems has been that the same nucleic acid sample is migrating differently in adjacent (or even the same) plug. Iíve checked and double checked for air bubbles under gel plates and I have not found any. (The ones Iíve found have been somewhat stubborn to remove though! Iíd appreciate any advice on removing the air bubbles that work their way right up next to the acrylamide squares in the bottom of the gel.)

I initially thought that leaving my acrylamide solution exposed to air over the 2 hour period of gel pouring may have resulted in dissolved oxygen re-entering the acrylamide mix (and possibly leading to changes in polymerization or polymerization rates). However, I got the same results even when I kept my acrylamide solution in a degassed environment over the entire time course that it took to pour the gel. My acrylamide solution contains 10mM Tris-Acetate, 0.1mM EDTA and 7.5% acrylamide (29:1). I add 70ul of 10% ammonium persulfate and 20ul of TEMED to every 10ml of acrylamide mix just before I pour the acrylamide. The gel is run in the cold room at 4C.

Right now, my inability to get the nucleic acids alone to run consistently has hampered my progress. Did you encounter problems of this sort when you ran PACE gels?

Thanks in advance for your time.

-- Rupinder Singh (rsb7@po.cwru.edu), November 15, 2001

Answers

I did not have problems with the same sample running differently in different wells. A couple of questions...

- Sample from the same tube runs differently in wells in the same plug?

- That tube isn't tampered with (heat/cool, etc.) between loadings?

- The same nucleic acid runs differently in plugs of the same concentration of protein/peptide?

You can expect to see a bit of "smiling" in the gel, depending on how fast you run. The heating of the gel isn't quite uniform across the width of the gel and so the edges run a bit 'slower' than the middle. But it never accounted for serious deviations in the overall migration patterns. I never ran a PACE gel in the cold room so I can't speak to the effects of that. There is no good reason why the same sample should run differently in adjacent wells of the same plug. How are they "different"?

As for bubbles... I took an old piece of X-ray film and cut a long strip with a "hook" on the end. You can slide it down between the plates and use the hook to move the bubble out of the plug right after it is poured. Also... Knocking the glass with a glass bottle (gently!) can help bubbles travel up to the top of the plug before it polymerizes. Using clean plates is the best defense against bubbles.

People run gel shift gels in the cold in an attempt to "maintain" the free and bound forms as they enter and travel through the gel. Since PACE maintains an "equilibrium" state throughout the run, you don't necessarily have to run it in the cold. If you are looking for temperature effects on binding, that's another story.

(Sorry this took a few days to answer, I was away on holiday.)

-- Christopher Cilley (cdcilley@scripps.edu), November 29, 2001.


Moderation questions? read the FAQ