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In gel electrophoresis, what causes effects like these (see collumn 11 in the first one, an collumn 6 in the second).

this this?

(These images were samples that I took from an online activity we did for class)



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up vote 8 down vote accepted

These are sequencing gels (in the cases here even radioactive ones) - they are run a lot longer than ordinary agarose gels and they are made from polyacrylamide. Im my experience, the most likely cause for skewing of lanes (not only bands) are samples, which still contain too much salts from the PCR reactions. This can also happen to only a few bands as seen here. Overall these are still pretty nice and clean gels, I have seen much worse.

Other reasons which cause uneven gels are cases where the acrylamide is not mixed good enough (causing the gel to have different concentration and thus causing different running conditions), uneven warming (although this was usually preventing by the gel heating system) and leaky gels where the electric field was not completely even.

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Could please elaborate on why too much salt from PCR can lead to this ? Thanks! – biogirl Feb 27 '14 at 8:07
The different salt concentrations cause different conditions for the currents which flow through the gel. When the conditions are not uniform, then the field is not uniform, which causes the transported nucleic acids to not move uniform. We could call it a "disturbance in the force" :-) – Chris Feb 27 '14 at 8:19

@Chris's suggestion is very possible, high salt characteristically shows this towards the end of the gel. But there are additional suspects. This looks like it's just an agarose gel, Correct? I've seen a few things cause this including the gel not being level, the gel shifting during during it's run the percentage of agarose not being uniform and the temperature jacking up towards the end. If you do it again keep an eye on your amperage during the second hour or so.

But first, try doing a quick PCR cleanup on a column (be sure to get one that will not loose your smaller fragments) or alternatively a phenol extraction followed by an ETOH precipitation will purify your reaction; and the force will be with you.

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