We have been having consistently anomalous migration of high / higher MW DNA on our agarose gels. When running a linearized 5.3 kb plasmid, it's running above the 10 kb MW standard. However, 'mid-range' DNA [i.e., 500 bp to 1,500 bp] seems to migrate where it should. Our gels are typically 0.8-1.0% molecular biology grade agarose in 1X TAE. We've tried different markers, different restriction digests / band sizes, and at 25 vs. 50 vs. 75 vs. 100 volts. One of the few variables we haven't isolated is our water, which is regular dH2O from our chemistry department [not nanopure as I used in my previous lab]. Any thoughts or suggestions? Thank you!
Hi Dina, normally there are two parameters that influence nucleic acid migration in an electric field, pH and salt concentration, since they affect the current going through the gel as well as the charge state of the nucleic acid.
So my suggestion is to check pH of your TAE and verify that it is around 8.3 and not lower, otherwise DNA will be less negatively charged and migrate more slowly.
Secondly, normally one uses TAE 50x stocks. Make sure the 1x is really 1x... possibly somebody diluted 1:100. Just make a new dilution making sure it's 1:50.
Third, check your 6x DNA loading buffer. There is not much in there, but, as for the running buffer, it should be at least at pH 8.
Hope this helps...
Are you sure that the enzyme is digesting your plasmid properly. If the enzyme you are using to linearize the plasmid is bad, you may not be digesting the plasmid and what you are observing is the open circular version of the undigested vector. Nicked, open circular plasmid DNA often migrates much higher in the gel relative to supercoiled or cut.
Thank you for this! The pH of our TAE is on the low side [8.15] so that may indeed be the culprit. I'll try a side-by-side comparison next.
Mmmmh, pH only a 0.15 lower, I don't think that's enough to justify the slow migration.
I would check the the salt concentration, make new TAE 50x... Somebody might have made a different stock concentration, confusing with some other buffer, and forgot to note it, or who knows what... It's easy enough, for 1 L just weigh 242 g of Tris (Tris base, or Trisma) solubilize in 750 ml of dH2O and add 57.1 ml of glacial acetic acid and 100 ml of 0.5 M EDTA, mix well and bring to volume...
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