i use the same reagents that my labmates use, but i have this queer problem killing me: after mixing the resolving gel ingredients (d.water, tris, acryl, aps, temed), when i pour the gel to the glass setting, and overlay the gel (isopropanol/ethanol/n-butanol-100%) sometimes it seems as if the alcohol has found the way through across the gel, making a river like shape. Sometimes the resolving gel has some horizontal streaks, or some curves on random places, or cloud's lining down the bottom of the gel, or a dripping water like shapes at the top of the resolving gel from where i pour the gel (which is the middle of the glass plate/ i have tried pouring from a corner of the glass plate-spacer), nothing works. I tried gentle mixing, vortexing, or vigorous mixing of the ingredients, still no luck ! Hope you got the explanation of the problem. I tried to take the pictures, but the lines are not visible, only visible when looked through a source of light or near window. This makes my gel wavy and uneven shaped. and my actin also fuses but i guess that's because of my slow loading. Apologies for the length of my question. Thanks !
Hi Ray,
I will advice you to take a proper care when it comes to gel setting ,i think you should properly clean you glass plates and pour you resolving gel by pipetting into the glass plates.Ensure that the glass plates doesn't have excessive pressure applied when placed on clamp assembly.Overlay separating gel with water carefully(not too much)and slowly.Try water and observe first,you will discover that the mixture gradually rise till you have an evenly distributed leverage.Do it in a different way than your routine.
I think this will help you
All the best!
Pouring PAGE gels can sometimes be a nightmare. As already mentioned, make sure your glass plates are absolutely clean before poring the gel. A good practice is to wash them as soon as you remove the gel so that the polyacrylamide does not build up on the plates.
Also, a quick hint that may be useful, you can overlay the resolving gel with the stacking gel without having to overlay your resolving gel with water, or any type of ethanol etc... Just carefully overlay the stacking gell over the resolving gel the same way you would overlay your H2O/ethanol (you may see a little bit of mixing, but this will not alter your gel). This process should be a litte easier, and most importantly save you time in waiting for the resolving gel to solidify, then waiting for the stacking gel to solidify.
- I completely agree with Adesola: Make sure your glass plates are super clean
- In particular wash with a detrgent like alconox or decon; Then rinse with dd water; then 70%IMS and allow to dry by evaporation
- In addition, make sure your plates are clamped tightly so no residual leakage
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