Plasmides run higher than marker - (Jan/03/2013 )

Hello everybody,

my lab is struggeling with our Agarose Gel Electrophoresis. Somehow the markers were always well separated over the gel but the DNA bands are stuck far higher than they should run. So if we try to separate digested plasmides/DNA, we get the right numbers and estimately right spacing within the bands but the bands are not running in the right correlation to the markers.

We use Seakem Agarose 1%, heated in TAE buffer. Gel is stained with 5ul Gelred/100ul Agarose/TAE. The Plasmide fragment length spans from 50bp to 10kbp - with dropping separation efficency correlated to increasing fragment lengths. The fragments itself are stained with Fermentas Green buffer (From digestion) or 1x Loading dye.

We already tried fresh TAE buffer, new Agarose, different gel concentrations, different running conditions.

I have the feeling that our electrophoresis chamber is not working properly since separation always requires at least 75min at 130V and 400mA (but you can see a strong bubbeling at the electrode). One test gel run in a chamber of another lab showed nice separation.

So what do you think? Its caused by the buffer? By the chamber? Or by the power supply? Any ideas?

Thanks!!!

A couple of things to keep in mind:

<*>The height of the buffer in the chamber. Though you are putting in a specific voltage and amperage more buffer will increase resistance, and therefore take away from the speed at which your bands run. Looking from the side of the box 0.5-1.0 cm above the cell is usually what I run. Not more. Plus, try to keep it consistent from gel-to-gel.
<*>The initial run speed. Most people ignore this, but it's a very useful trick. If I'm running a 120V gel, I always run it at 90V until the sample is all the way out of the well and into the lane (this is usually ~10 minutes). This way I can be more confident that my samples are running at the same velocity through the well when it counts. Basically, this is allowing your sample more time to snake its way into the pores of the gel, rather than cramming it in.
<*>Clean the electrodes in the box (that interface with buffer) with some diluted acetic acid or soap.
<*>This one is obvious, but make sure you're using 1X buffer.

If I think of others, I'll come back.

Dear JYaron,

thank you for your suggestions!

There is a filling mark in the chamber which is around 1-2cm above gel surface - do you think this might be too much? Nevertheless I often run with less buffer and this didn´t changed anything.

But the initial speed and the cleaning I´ll try! (buffer is for sure 1x )

Might there be some correlation to the temperature of the buffer? It really get warm during the 1 hour of running.

Heat up your Marker and cool it down. For the first time take an aliquot and heat up 94c. Use it look what happens.

The salt content of your samples can have a large effect. Also, assuming you are using horizontal gels, the buffer need only barely cover the gel. More buffer just leads to more heating and problems.