Uneven leveling in resolving gel - SDS-PAGE resolving gel woes (Sep/17/2009 )

I am a noob here. The problem is that whenever I pour a 10% resolving gel onto the BIORAD SDS-PAGE apparatus (1.0mm plate and comb), the gel is never leveled straight and looks kind of bumpy. I pour a little water on top of the gel to help polymerization and I mix all the ingredients correctly. I also follow a gel recipe chart that everyone in my lab is comfortable with. Still, I am not able to figure out where I am going wrong. Can anyone help me out here?

Kamal

Try overlaying with water saturated butanol and make sure you clean your plates well with water and ethanol.

bob1 on Sep 20 2009, 07:13 PM said:

Is it ok if I substitute water saturated butanol with water saturated isopropanol? And does the alcohol actually help to level the gel or does it only get rid of the bubbles?

No, water is miscible with propanol but not with butanol - if you use propanol you will dehydrate your gel and it won't fix your leveling problems.

bob1 on Sep 23 2009, 06:17 PM said:

I tried what you suggested. I clean the plates thoroughly with ddH20 and ethanol. I then tried overlaying the gel with water saturated butanol. Though at first it appears as if the gel is fine, once the gel polymerizes the leveling is awkward.

Am I doing something wrong while pouring the gel? Should I overlay the water saturated butanol slowly? Or is my gel recipe not quite right for the 10% gel? Any help is greatly appreciated. I am going crazy trying to figure out where I am going wrong. I use the Bio-Rad mini protean cell setup to cast my gel.

I had experienced that before. Maybe you should be more careful when you overlay your gel with water and/or butanol and distribute the solvent evenly. Water is fine with me. Also, check the amount of APS/ TEMED. High amount of these can lead to rapid polymerization which may lead to uneven gel since the gel may have already started polymerizing even while you are still pouring it on to the cast.

I know isopropanol is soluble in water...but that's what I (and all of our lab) has been using to overlay the resolving gel, with no problems. Maybe the high solute concentration in the water prevents mixing...also pipette the alcohol gently (I usually do pipette into the walls of the glass plate) to minimize disturbance of the acrylamide solution. Hope that helps!