I have been working on making Polyacrylamide gels from 30% Acrylamide and 2% bisacryalmide stock solutions. I prepare the polymer solutions in a centrifuge rube, add APS and TEMED to the solution, do a quick vortex and then pipette out a few micro liters onto aminosilaniized glass substrates slide. I place a hydrophobic coverslip on top and leave the solution for 40 mins. The solution is not polymerizing completely and I am getting a gradient of stiffness in the gel (as opposed to a uniform stiffness). I prepare the APS fresh everytime. Any idea why this could be happening?
I had once left the bisacrylamide solution at room temperature (light proof bottle) over night to ensure the bisacrylamide completely dissolves. Could this be a reason?
Dear , at first look you forgot degasing, oxygen is an inhibitor/retarder of radical polymerization. My Regards
Um, good point but no one in my lab has been degassing it and the gels worked fine for them. I assumed a 1mL solution volume is too small and degassing wouldn't matter. Maybe, I am wrong. I do not have a degassing chamber on the lab. Any ideas how to go about the degassing?
Deagasing is simply done by passing a flow of an innert gas such as N2 for a période of time say from 15-30min and making a blacket of it. Other possibility use a freeze-thaw cycling.
Best of Luck
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