Troubles with casting Gradient Gels - (Dec/02/2011 )
I'm having problems trying to cast 4-13% polyacrylamide gradient gels for BN-PAGE. I'm using a gradient mixer for the acrylamide solutions with a peristaltic pump in a cold room when casting... recently the gels have been showing like a tear-drop shape rising from the bottom where the different percentages are not setting in a linear fashion. I don't think it is air bubbles as it seems to be just the acrylamide rising through the centre of the gels... All my solutions and equipment are chilled before adding to the gradient mixer and I've meticulously cleaned the glass plates. I'm stumped!
Any help would be greatly appreciated!!
why are you pouring the gel cold?
cold will trap gasses which will inhibit polymerization. you should be pouring the gel at room temperature.
the shapes may be caused by the heat generated by the polymerization reaction (convection, release of trapped gas).
All of the protocols I have looked at for pouring these gels have recommended using cold equipment and solutions to prevent the gel setting in the pump.
But after reducing the TEMED and APS and using the pump at room temperature I have managed to get non-distorted gels!
Thanks for your help