tricin gel problem - shrinking of stacking gel (Dec/12/2005 )

I am facing a problem in Tricin SDS PAGE, during solidification of Stacking gel the wells become very small due to shrinkage (even resulting in irregularly formed gel). Please suggest a solution and reason for this effect.

Reagent composition for Tricin SDS PAGE Stacking
Acrylamide-Bisacrylamide Mix(49.5%T,3%C) = 400 microliter
Gel buffer = 1.24 mL
Water = 3.36 mL
APS = 40 microliter
TEMED = 4 microliter

Thanks a lot

Rick

check the pH of your buffer?

hi aimikins
ph of my gel buffer is 8.45 ?? any suggestions..??

avoid the gel meet air and check if the tomb has broken

CHECK WHETHER THE TIP OF YOUR WELLS ARE IN CONTACT WITH AIR.

SECONDLY ALSO CHECK THE IF THE WELL TIPS ARE UNIFORM FOR THEY MAY LEAD TO CONTACT WITH AIR WHILE MAY NOT BE KNOWING IT.

GOOD LUCK
GAGAN

do you use fresh APS?
your protocol seems OK.

i don't know how long you're letting this polymerize, but given the concentrations of acrylamide:bis, as well as the amount of APS/TEMED you're using, if you let it polymerize for more than an hour or two your gel will start to pull away from the wells.

try decreasing the amount of time to polymerize. the gel doesn't need to be like a piece of plastic to be finished. if a small amount of unpolymerized acrylamide/buffer remains in the wells after an hour or so, just rinse it out with water, and cathode buffer w/ a syringe prior to use.

good luck

.