Protocol Online logo
Top : Forum Archives: : Molecular Biology

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


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.


Josh Attached File


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.



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.


Reviews of DNA sequencing services

-Daniel Tillett-