how to make nondenaturing gradient gel - (Feb/17/2012 )

I have many problems about making nondenaturing gradient gel. I try to make 2-16% nondenaturing gradient gel (without SDS) but I don't know how to do it. Can someone please help me?

1. How many volumes of solution A (16%) and B (2%)? Do I have to fill them in the reservoir as eual volume? (I use Hoefer miniVE 10*10.5 cm)

2. When do I open the valve of solution A and the valve between A and B? In my opinion, first I have to fill the solution A and B in reservoir, then open valve A (how long?), open the pump, finally open the valve between A and B (there is the flow of solution A and B in this step). but I'm not sure what the right protocol is.

I hope someone can help me...

1. equal volumes of solutions A and B, respectively

2. I use to open both valves simultaneously; use a stirer for tube with solution A; I do not use a pump but flow by gravity

we use unequal volumes due to density differences (which we enhance with sucrose, also different percentages in the heavy and light solutions). the exact volumes depend on those densities.

we always allow the heavy solution to completely traverse the bottom before opening the valve between solutions (we pour from the top, if you pour from the bottom then you will want the light solution to go first).