I've been doing some PAGE gels recently on PCR products, and every so often the front gel comes out smeared (including the ladder). I'm using the same reagents and procedure. It's not my technique as the back gel is fine, and other people in the lab have tried doing it, with the same results. What could cause this problem in the ladder as well as the samples, on just the one gel? I'm thinking maybe there is a problem with the tank or the powerpack - perhaps the voltage is too high across the front gel on occasion? My manager says it could also be the time it takes to load, but it's only an intermittent problem, so I'm not convinced. There doesn't seem to be any pattern to it.
I guess you have one of those PAGE chambers that allows you to run two gels at the same time and if you run just one, you need to use a "dummy gel"?
For me it sounds like a problem with the buffer reservoir on one side. The gel on one side is not fastened tight enough to the apparatus and the two buffers have a direct connection. Seal it more tightly, vacuum grease for centrifuge rotors for example is a good thing for the sealing rubbers.
The loading time should not be the problem since the bands are focussed on the stacking part of the gel during the run. A too long loading time results in complete loss of sample if it diffuses out of the loading pocket into the buffer.
At what voltage and for how long do you run ur gel? Try to run you gel in cold room or keeping the tank in an ice buket. Other possible reason could be altered pH in your buffers used for casting the gels. As I had similar problem, and after making fresh buffers my problem was resolved. Hope it helps.
Thanks Vivica and Gopal for your answers. It is a two gel chambered tank. I'll look into your suggestions! The gel is run initially at 150V until the samples enter, then at 300V for up to 1 hr, or until the samples are at the bottom of the gel. We make up the buffer for making the gel fresh every time, from 10x stock, and the running buffer is made up fresh every few days (we never re-use buffer for these types of gel). I should say that we don't have a stacking part of the gel, it's all one type of gel.
I've recently tried using just the back position of the tank, which seems to have alleviated the problem (though not ideal). So it literally is the front position that is the problem :(
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