gel electrophoresis of DNA : failed!



  • Hello,

    Yesterday I ran a gel electrophoresis of some DNA samples with a ladder. Normally, I look at the colour of the dye and eyeball it to decide when I should stop running the electrophoresis. When the dye is about halfway down the gel, that's when I usually stop. With yesterday's sample, I decided to try a new PCR buffer that already has a GoTaq loading dye included. The gel ran, but it took almost TWO HOURS for the dye to get even just 45% of the way down (I used 125V too, the product size was only about 300bp)!! The really weird part was when I imaged the gel, the band that I was looking for was present, but it was almost at the VERY BOTTOM of the gel (about 90% of the way down). This was a new phenomenon for me because I normally found the band at the same area where the dye was (and you can see this with the naked eye on the gel). Instead, it was found nearly double the distance down the gel. What's the deal with that?

    I'm running another gel right now with a new PCR product (same gene, same sample), but without the special buffer with the dye included. I'm using the regular buffer and loaded my own dye in before I ran the gel. I also put in the sample from yesterday to see how it runs again. I've set it on the same voltage and hoping to see what happens.

    Another funny thing I noticed yesterday - the current was almost at 500IA!! I've never seen it this high before. The voltage was still set to what I had set originally, but the current was off the charts. I had actually forgotten to unplug the other container connected to the machine (so the electricity was being divided into two), and I assume this was the reason why the current was so high. What do you think guys?
    Please Help.

    Thanks !

    I didn't find the right solution from the internet.

    References:

    http://molecularbiology.forums.biotechniques.com/viewtopic.php?f=2&t=32548

    B2b Digital Marketing Campaign



  • @brentmarble3

    Hi, I read through your message and I'm wondering whether this is a general question about gel electrophoresis or whether you are having trouble with your electrophoresis equipment. Are you using our IO Rodeo gel box and electrophoresis power supply ? It doesn't seem from your message to be an equipment problem, but more a problem with a new loading dye that you are using ? If that is the case, you might be better off asking the manufacturer of the loading dye whether you should expect to see you DNA sample behave differently in their buffer. I'm sorry I haven't used the GoTaq loading dye.
    I hope that helps. Good luck with your PCR experiments and let us know if you have any specific questions about our electrophoresis equipment.
    Jo


Log in to reply