Acrylamide stock for gels - which buffer (May/29/2006 )
Hey, I was given an acrylamide powder as a gift/for free and to use up before it expires. I usually used acrylamide and bisacrylamide solutions in my old lab for my acrylamide-gels. Now my question is in what do I dissolve the powder and what do I use instead of bisacrylamide.
This was my old recipe for my gels:
Resolv. Gel (12 %)
4.5 ml 40 % Acrylamide
2.4 ml 2 % Bisacrylamide
3.75 ml 1.5 M Tris HCl
175 µl 10 % SDS
75 µl 10 % APS
75 µl 10 % TEMED
4.2 ml H2O
Stacking Gel (4%)
500 µl 40 % Acrylamide
260 µl 2 % Bisacrylamide
1.25 ml 1.5 M Tris HCl
50 µl 10 % SDS
25 µl 10 % APS
5 µl 10 % TEMED
2.9 ml H2O
Now how do I manage this with my super-free powdery acrylamide???
Dissolve 400g acrylamide in 1L of milliQ water to make 40% acrylamide.
Then filter through Whatman #1 paper to remove undissolved powder.
Store at 4 degree in brown bottle or normal reagent bottle covered in foil.
Hope this may help.
You can also recrystalize "expired" arcylamide for greater purity - dissolve 70g in 1 L of chloroform (as pure as you can get by resonable price), heat to 50degC (fume hood!), filter through Whatman and chill to -20degC. Decant, wash again with chilled chloroform and dry.
So I will just dissolve it in Millipore water??? That's all???
dissolve and filter. yes. that is all.
also, use your bisacrylamide as in your formula.
Thanks guys, this is almost sounding too simple Yayh, finally at least something si simple.
Still I'm confused about the bisacrylamide, I only have normal acrylamide powder, no bisacrylamide. So will I just leave it out or how to I get the "bis" in my acrylamide
according to the formulae in your earlier post you add bis separately. just continue to do it the way you have been, as a separate solution.