Biology Forums - Study Force

I tried to do an SDS-PAGE gel but after pouring the separation gel, this is what I got:

(http://s2.postimg.org/55jjjldxh/photo.jpg) (http://postimg.org/image/55jjjldxh/)


When I poured the gel, I made sure that there were no air bubbles whatsoever and the top was a very smooth line. No air bubbles developed during polymerization either but for some reason it polymerized irregularly! What I've noticed is that 2-3 mm at the very top, there was a liquid layer, up to the bottom marker line (the image was taken after I poured out the liquid that remained to make the gel surface more visible for the pic). I'm sure I left it to stand enough because I left it for like 30 mins.

I tried doing another gel and adding water on top of it...I did get a smooth surface but the front was obtained very low down the plate as if the water I added diluted the first few millimeters of the gel and so the first 1 cm from the top didn't polymerize.

Any ideas? I've tried 2 recipes and got the same results when it comes to surface 'smoothness'.

How are you adding the water? The water should be pored very slowly from sides.

The picture attached is of the gel I casted without adding water. As I said, the surface was smooth without air bubbles until it polymerized as such. The one in which I added water, I added it using a pipette onto the glass to prevent mixing.

The acrylamide wont polymerize in the presence of Oxygen. It is important to avoid the contact of gel with the air during polymerization. Instead of water you can use isopropanol to cover the surface of polymerizing gel. In our lab we used to degas the Acrylamide mix using vacuum before adding APS and TEMED. Degasing step allows better polymerization and less chances of uneven surface..

Degassing using nitrogen?

Not nitrogen... Just shake the flask under vacuum. This will remove all the air from the solution.

Oh ok. I'll try that! What are the advantages of using isopropanol instead of water? Less miscible? Is it normal that SDS gets 'foamy'? I know technically its 'soap' but even when mixing it forms quite some bubbles (both as 10% and 20%).

oxygen is relatively less soluble in Isopropanol  than water. Also Poly-acrylamide is less soluble in alochol too so it wont dilute the solution on the surface. It will draw some water from the gel though but should not affect polymerization. If you worry, use alcohol and water mix (50%). If you degas your solution well, water  will do fine as air barrier.

Foam in SDS solution is normal. It will happen no matter how much careful you are. Make little extra solution ( I am sure you are already doing this)  than you need so you don't have to pour bubbles. Always pickup from the bottom of the container and don't push everything out while pouring between the glass.

I'll give that a shot :) Cheers!