Yuhsaku Godai [Ronin] wrote:
>> I'm trying to run my first SDS-PAGE gel but can't seem to get the gel
> solution correct. From the Molecular Cloning lab manual, I've been
> trying to make 5 ml of a 10% gel but it's not polymerizing completely.
> Although addition of either more ammonium persulfate or TEMED seems to
> make it gel properly, why isn't the amounts given in the manual correct?
> Are there other factors I need to take into consideration? Any help
> appreciated. Thanks.
I checked what is in Maniatis with our protocol (used a long time w/o
problems) - they basically match, so it's not the recipe. I didn't read
the whole Maniatis thing about gels...are you degassing the gel mixture
before polymerizing? Oxygen will quench the reaction...also the problem
if you're running a very small or thin gel...the acrylamide solution at
the very top will be less likely to polymerize since it's in contact with
the air. One thing we routinely do is make up more gel solution than we
need and pour in a little extra volume for this reason. BTW, the recipe
in the book comes out to 4.9 ml, not 5 ml. I can't think of too much
else than the degassing thing...the only other thing would be to try
fresh APS if you're not doing so already. My boss swears by it and won't
let us re-use it once made.