northern blotting - (Oct/17/2001 )
I run the Agarose/Formaldehyde gel at 45 V during 5-6 hours. After the exposure I obtein thick bands. How can I improve the quality and resolution of the bands? Should I use more volts in the electrophoresis step?
A good way to find out is by simply trying. Ofcourse this is RNA you're working with and I only have experience with DNA electrophoresis. With that, 100 Volts for 1 hour or so is very good useable. I think a somewhat higher Voltage would be better, including things like a less dense gell.
If you are getting very thick bands, this could be the result of a few things....1. your wells are very wide ; if case, use a different comb when the gel is solidifying2. you are loading too much sample; prior to loading the RNA in the well, I am sure you ran a 260/280 in orderto determine OD which (after conversion with equation) will equate to RNA concentration. if you see yourbands are too thick, simply lower the concentration that you load.
I personally do not believe that altering the voltage at which you run it will result in more concise bands.I would first spread the sample out into a few wells. what's the prob. with such wide bands. are you extractingfrom the gel?