Problems with wells on SDS PAGE gels - (Mar/11/2014 )
I'm having a weird problem that no one at work has been able to figure out. I've been making gels with 10% resolving gel and 5% stacking. I use the appropriate sized comb for the plates that I'm using, yet every single time I pull the comb out the wells are filled with bits of gel. It's almost like there's a gel membrane tha runs across the top of the well and some globs of gel in the bottom. This makes loading samples near impossible, even after I have flushed the wells out with air. I've been lettting the stacking gel set for 15-30 minutes. It's has definitely set by the time I remove the comb.
Anyone have any suggestions? Could this be cause by being sloppy when removing the comb? Should I let it set longer?
Check your stacking gel composition, if you have too much APS or TEMED you can get a very sticky gel.
Make sure when polymerizing that the whole comb bit is set, such that the edges are sharp, not rounded (assuming you are using a normal square tooth comb).
Wash wells out with water or running buffer rather than air. I like to use a squirt bottle for this, and squirt into each well for a couple of seconds each, then flick/shake solution out. Repeat if necessary
one possibility, when the stacking gel solution overflows during insertion of the comb you may have some polymerize on the edge of the plate. when removing the comb this will get disrupted and suction will pull it into the well. you may have to remove it by scraping it out with a spatula.
another possibility, your plate may have warped. this allows a void to form between the comb tooth ant the plate where stacking gel can polymerize. again, this "skin" can be removed with a spatula and flushing.
Hi everyone, thanks for the replies. Sorry for the late response. After talking to some labmates, someone suggested that perhaps it was the layer I was putting on top of the resolving gel while waiting for it to set. The lab that trained me uses 70% ethanol while waiting for the gel to polymerize. Someone told me that they had never heard of using EtOH and that most people use butanol. I tried using butanol on two different gels last week and they both turned out perfectly, no need to even clean out the wells. I just made sure to throughly wash the butanol off with water, then pour the stacking gel. Perhaps the 70% EtOH was reacting with the stacking gel somehow and made it sticky.
I figured I'd mention that in case anyone else has the same problem.