So I ran into an interesting problem. I'm getting a linear DNA band that is twice as long (4x bases, but as denatured probably only 2x) as an RNA band running at the same size in a formaldehyde gel.

Both sequences have been isolated an 100% confirmed. The gel was run in MOPS buffer. My experience, and all the publications I've found, show that if anything RNA should run faster. Can anyone think of a reason why the DNA band and RNA band would be running the distance despite the DNA being twice as long?

I've read a lot of fun papers on drag factors in gel matrices now, but none of it leads to this confusing and repeated result.

Edit: Gel and Conditions

Gel was 1M formaldehyde, 1% agarose. Run at 100V for 1.5h. RNA was mixed in a RNA loading dye which contained 15.3% v/v formaldehyde, 41.3% v/v di formamide, 4.6mM EDTA, MOPS, and bromophenol blue. We believe our RNA loading dye to be denaturing, and it ran at expected size with RNA ladder.


enter image description here

Lane 1 is a DNA template plasmid, cut 1 location.

Lane 2 is the Template + DNase from in vitro transcription reaction w/o polymerase.

Lane 3 is the RNA + DNase from in vitro transcription reaction.

There are no other bands in the DNA lane, and it looks like if anything, the DNA is migrating much faster than expected, despite being sequence confirmed (both before the process, and gel purified out and confirmed again).

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    $\begingroup$ I've deleted my answer as it was useless. In the comments you said that you're running a denaturing gel. Have you considered polyacrylamide/ polyacrylamide-urea gel? $\endgroup$ – Nandor Poka Mar 11 '15 at 19:48
  • $\begingroup$ Interesting. Do you have a picture of the gel? $\endgroup$ – WYSIWYG Mar 11 '15 at 21:04
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    $\begingroup$ I'm trying to get an OK from the tech office to post an image now. $\endgroup$ – Atl LED Mar 11 '15 at 21:10
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    $\begingroup$ Maybe your RNA does secondary structures... $\endgroup$ – Pao Mar 11 '15 at 23:59
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    $\begingroup$ Then off the top of my head, maybe the RNA isn't being fully denatured for some reason. That lane also looks very overloaded, could that alter the migration rate? $\endgroup$ – user137 Mar 13 '15 at 14:36

The inclusion of formaldehyde in the gel and buffer is to keep the RNA denatured (ie after heating the sample to melt the double-stranded stem-loops, just prior to loading the gel), in the hope that the RNA will migrate through the gel with an Rf proportional to its molecular weight (approximated here by its length). Therefore the correct size markers would also be made of RNA (and prepared the same way, by heating). It is difficult to predict, a priori, how a double-stranded DNA fragment would migrate under similar conditions, without also including a lane of ds DNA size markers. In other words I would only use DNA markers for DNA and RNA markers for RNA. By repeating this experiment (1 lane of DNA MW vs 1 lane of RNA MW) at different voltage gradients and different concentrations of agarose, you might be able to plot two standard curves that would let you predict where your DNA fragment is expected to migrate relative to the RNA transcript, but without that data I would not assume that a larger DNA fragment would run slower than its RNA transcript. Even if the dsDNA was exactly the same length as the ssRNA (and pretending that the MW of the dNTPs and NTPs are identical), the DNA's MW would be twice as much as the RNA, but the negative charge on the DNA would also be twice as much.

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    $\begingroup$ Instead of a standard curve, why not just run two ladders on your gel? 1 DNA and 1 RNA. $\endgroup$ – user137 Mar 23 '15 at 15:33
  • $\begingroup$ @user137 Exactly, that is the immediate practical solution. $\endgroup$ – mdperry Mar 23 '15 at 16:46
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    $\begingroup$ While I agree with everything you mentioned, it doesn't really address the issue. I've been working on a standard curve. It is odd that the DNA is migrating much quicker than expected, as you point out it should be slower for the same length, doubly so for twice the length. And yet under multiple conditions I am seeing the same thing. Might be the long poly-A tracks in the DNA, I don't know. At this point it's not as import for the project as it is a curiosity. $\endgroup$ – Atl LED Mar 26 '15 at 16:48

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