Making those stupid gels is so tedious and pain-staking. Me, the new guy, has epically failed the first 16 times making them. Does anyone know what I am talking about here?
I freaking hate polyacrylamide gels
- Thread starter UCFMOP
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Clean both plates with alcohol. Then with hydroxy methanol solution. Than siliconize the top plate for non-stickiness appropriately (plate with the cut-out for the comb).
Lay the bottom plate absolutely flat (use level) with spacers on three edges to create a shallow reservoir. Put the comb at the top. Add the TEMED to begin the cross-linking reaction. Pour immediately along the bottom spacer. Keep a bit of the activated acrylamide solution in the fridge.
Take the top glass plate and touch the bottom of its edge to the bottom plate's bottom edge. Slowly begin to move the top plate down and the acrylamide will slowly edge towards the comb. Go slowly and the gel will not form any bubbles. Once in place, clamp all edges. In 5-7 minutes take the remaining acrylamide out of the fridge and pour near the comb. Capillary action will suck it into the plates and relieve any gaps created by the polymerization process.
Good luck.
Lay the bottom plate absolutely flat (use level) with spacers on three edges to create a shallow reservoir. Put the comb at the top. Add the TEMED to begin the cross-linking reaction. Pour immediately along the bottom spacer. Keep a bit of the activated acrylamide solution in the fridge.
Take the top glass plate and touch the bottom of its edge to the bottom plate's bottom edge. Slowly begin to move the top plate down and the acrylamide will slowly edge towards the comb. Go slowly and the gel will not form any bubbles. Once in place, clamp all edges. In 5-7 minutes take the remaining acrylamide out of the fridge and pour near the comb. Capillary action will suck it into the plates and relieve any gaps created by the polymerization process.
Good luck.
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Ugh, I know exactly what you're talking about. They are so finicky. I had to run quite a few of these in my undergrad lab - but not quite enough to actually get good at them, of course. Buying them sounds like a good option, for sure! 👍
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Loving the talk in this thread. Most real labs buy premade gels. Guess I didn't work in a real lab for that year after undergrad.
yesss, i know. they're like $8 a gel!!! we can't afford that!
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mine used to leak all the time. If you can use parafilm or lab tape to seal off the bottom of the gel, try that.
only 1 lab I worked in bought premade gels. i didnt like them because they were a pain to crack open.
only 1 lab I worked in bought premade gels. i didnt like them because they were a pain to crack open.
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Wow I'm so jealous. I had to make my own even when I had to do 6/day. 🙁
Btw did any of you ever have experience with having your bare skin exposed to EtBr?
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sorry didn't mean it that way. i thought most labs moved on to premades now since they are relatively cheap (less than $10 each)No, did you? please share. i had a little DMSO (dimethyl sulfoxide) on my hand once, i didn't notice it right away. It felt like my entire hand went slightly cold. according to wikipedia it penetrates the skin easily and you should be able to get a garlic/oyster-like TASTE soon. i did not get this but I am curious to try it one of these days.
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I made a few for the first time last week. The first two I made failed because they leaked. Apparently,the trick is to make certain that the two plates are perfectly even at the bottom. You can scrape your fingernail over the bottom and check that you don't feel a bump as you scrape from one plate edge to the other. Also, *lightly* place the plates into the gel casting device - don't push them into the gummy/rubbery bottom, since that will actually cause leaks. You can test for leaks by putting some 100% EtOH between the plates. You can then dump out the EtOH by turning the casting system to the side and letting the EtOH run off into the sink/pipetting the remainder out.
As for the actual casting, I just followed a recipe and it seemed to work. After the first two gels failed, I made four more and ran them successfully. Add butanol on top of the resolving gel as its setting to make an even interface. Wash the wells out with ddH2O before loading.
As for the actual casting, I just followed a recipe and it seemed to work. After the first two gels failed, I made four more and ran them successfully. Add butanol on top of the resolving gel as its setting to make an even interface. Wash the wells out with ddH2O before loading.
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If you're using plates with spacers glued onto them, make sure the butanol doesn't wash away the adhesive. Otherwise, you can gently add water instead. It works. I've done it more times than I'd care to remember.
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