High MW bands are ugly - electrophoresis (Feb/20/2006 )

Hi,

I have been using 1% TAE gels for DNA, and my high-weight bands often (almost always) have
a very tattered look to them. This effect is seen also with the ladder (in the example, the left-most lane is promega's lambda/hindIII). As you can see, the 9 kb and the 23 kb bands look like they are trailing shredded garbage behind them. This gel was run at 100V (~7.5 V/cm) for 1:40 and then stained in buffer/ETBR for 1 hr.

Thanks,

Josh

It looks like you might be overloading the each well with too much dna. How much of DNA is loaded for each lane?

You are overloading the gel.

-Matt

This seems to be the consensus. I'm not sure about my samples, but the lambda standard being loaded is 1500ng total.

These are plasmid RFLPs so I need to see bands below 1 KB as well as 20 KB+. It seems difficult to load just enough to see the low MW bands, but not so much as to overload the high MW bands.

Why not load 2 lanes then? One with a lot of DNA so you can visualise the 1 kb band, and another one with less DNA to visualise the 20 kb+ band?

Assuming they are present in equimolar amounts, you'll have a 20-fold difference in total DNA present on the 20 vs 1 kb bands so it's hard to visualise the smaller one without having overloaded the other.

Or use a marker with equal amounts of DNA at each band- NEB sells a few. It also helps to run you gels a little slower too.

Daniel

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