I am working on denaturing Urea-PAGE. But after the loading of DNA samples, the bands showed wavy or slope pattern instead of straight. What would be the reason? Is there any reason based on PAGE concentration (i.e. Acrylamide-Bisacrylamide concentration)? I have used 4% and 6% PAGE with the ratio of 19:1 for Acrylamide : Bisacrylamide.
Also please tell me how can I clean my PAGE glass plates from urea solution as urea makes PAGE plate turbid after sticking to it. It takes lot of time to clean.
Thanks.
Hi there,
To get an even run you have to preheat the gel, you may also apply a metal plate onto the glass plate to uniformly diffuse the heat. Urea being very soluble in water plate washing is pretty easy...
To add to before you want to make sure that you flush out the wells before loading your sample, residual unpolymerised acrylamide and urea can affect the running on the gel. So I would flush wells gently with a needle and syringe, followed by running the gel for approximately 10 mins at 100-200V depending on the gel size. This will prevent smiling. The other option is running the gel at a lower voltage and therefore slower.
Cleaning just requires a lot of warm water and a gentle scrub, I find that even just a double gloved hand and rubbing off the crystalline solid is fine too.
Wavy pattern might be observed if you have some excessive salt in your loading samples. If there is no way to desalt your RNA preps prior PAGE, you can run electrophoresis at lower voltage (10-15 Volts per cm of gel length) as Simon suggested. This is not going to completely solve the problem but the bands might look better.
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