Help with SDS Gel Mixture

Anonymous anon at email.wherever
Fri Mar 15 02:39:40 EST 1996


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.
> 
> -Ben
> --

Dear Ben,

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.

Best regards.



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