Acrylamide Gel Problems

[molecGENE]

I was making an 40% (19:1) acrylamide:bis gel with TEMED and ammonium persulfate, and as a used a syringe to make the gel, the solution in the syringe polymerized, but the remaining in the beaker and the solution already between the plates was still fluid.

 

-How do I prevent this in the future?

[CharonY]

If only partial polymerization occurs it seems that your solution is not well mixed. First add TEMED, use a stirring bar or something else to mix really well, then add APS, allow for stirring, then pour.

[Max_Normal]

Are you using this for nucleic acid separation? I use agarose myself, but I thought that 6% to 12% was a normal range for a nucleic acid acrylamide gel.

 

I use acrylamide for protein, just 4% to 17.5% depending on what i want to see.

 

40% just seems very high percentage, I have never heard of it being used straight out of the bottle, have you checked your protocol?

Edited March 3, 2012 by Max_Normal

[CharonY]

I assumed that this was not the final concentration but the standard stock Although in hindsight that may have been an issue. 19:1 is typically used for denaturing nucleic acid gels. Although technically there is no fundamental reason against using it for protein analysis. The limiting factor in polymerization is usually the availability of free radicals, though.