The top of PAGE gel is not polymerizing - (Aug/23/2007 )
I am using the following recipe to prepare 60 ml of a 10% Polyacrylamide gel:
15 ml 40% Rotiphorese
33 ml Water
13 ml 5X TBE
60 ul TEMED
420 ul 10% fresh APS
The problem I have is that the liquid around the comb apparently is not polymerizing, so when I put the comb out I don't have the wells to put my samples. The strange thing is that the rest of the gel is OK.....the problem is only in the top of it.
Thank you for your help!
What is Rotiphorese? Is it the brand name of acrylamide?
Yes, it is the acrylamide
Most probably air is inhibiting polymerization. Try to degas your gel solution (before adding catalysts) and wrap top of the gel with parafilm or saran wrap.
during acrylamide polymerization, water is always released at the top. in fact, we use this phenomenon to see when the gel is polymerized (you see a second meniscus form under a butanol overlay).
you may be able to avoid this by allowing your gel to polymerize for a longer period of time.
or even overnight.
I second overnight polymerization - it works very good for protein SDS-PAGE gel.