I am running a 20% PAGE gel to analyze the formation of different DNA complexes with lenghts between 6nt and 40nt. The high gel concentration is necessary to achieve proper resolution. Lower gel concentrations have resulted in severely smeared bands. However when increasing the gel concentration to 20% the bands start to travel not only vertically but also horizontally. Resulting in the outer lanes leaving the gel. Can anyone explain to me what the reason for this might be?
The PAGE is run under the following conditions:
-Voltage: 170V
-runtime: 90min
-cooling system: ice bath
-Gel: 20% PAGE: 1xTBE +12,5mM MgCl2
-running buffer: 1x TBE
-samples: 5uM DNA complex, 0,5x TBE + 12,5mM MgCl2
I have just started doing PAGE experiments. I'm therefore thankful for any advice you can offer.
This looks to me like you have a leak in the gel box along the edges during the run, causing the electric field to bend outwards.
I think the explanation given by the previous responder is certainly valid. From my own experience, insufficient mixing of acrylamide or adding too much TEMED during gel preparation—causing the gel to polymerize too quickly—could also potentially result in such issues.
I would avoid using the two outer lanes on both sides, and initially run the samples into the gel at a low voltage, maybe 20V, then as soon as the sample dye has entered the gel, stop the gel and flush out the wells, then restart the gel and try running at 170 volts, though you may have to just run it at a lower voltage for the whole run, that or try running in a frig or cold room to keep the gel cold.
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