In my lab class this week my partner and I were making a SDS-PAGE Laemmli Gel. We made the resolving gel solution which was made of 30% Acrylamide, DI water, 1.5 M Tris-HCL pH8.8, and 20% SDS. We mixed the measured amounts into a conical vial. We then added 10% APS and TEMED and mixed carefully. We then immediately pipetted the solution into the casting stand so the gel could solidify. We then pipetted DI water onto the top of the gel so it would not dehydrate. We left some solution in the conical vial so we knew when the solution would be solidified completely. After waiting 45 minutes the gel in the conical vial was solidified but the gel in the casting stand was still liquid. How come the gel did not solidify in the casting stand but solidified in the conical vial?

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    $\begingroup$ How long is your polymerization time? How old are APS and TEMED? Why do you use water on top and not an alcohol? $\endgroup$ – Chris Apr 15 '15 at 15:36
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    $\begingroup$ You put a liquid on top of the gel because you want to avoid contact with the air. Air catches the free radicals needed for the reaction. Water can mix with your solution and dilute it (depending on the amount of water used and how cautious you pipetted it), thats why I usually use a few hundred microliters of isopropanol. $\endgroup$ – Chris Apr 15 '15 at 15:47
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    $\begingroup$ The polymerisation time appears to be rather long. From personal experience, it rarely takes more than 15 minutes for both the gel in the falcon tube and casting stand to solidify. How much APS and TEMED did you add? $\endgroup$ – March Ho Apr 15 '15 at 16:09
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    $\begingroup$ The water shouldn't be an issue if it's layered carefully. $\endgroup$ – canadianer Apr 15 '15 at 17:43
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    $\begingroup$ We added 150 ul of 10% APS and 15 ul of TEMED. The water was also added a couple drops at a time alternating between each end of the casting stand. $\endgroup$ – USF0101 Apr 15 '15 at 21:16