It depends on temperature. Polymerization will slow down, if the room temperature decreases. At room temperature, gel will polymerize in 30-45 mins. Freshly prepared APS is much necessary .
Hi,
Lower temperature will delay the polymerization and not accelerate/fasten it. So if you are not used to cast gels quick enough, then it I'd suggest you to use bit cooler solutions (~15C). Such a gel will take ~1h to polymerize.
Acrylamide gel polymerization depend mainly of the temperature. Normally, it takes around 40 min to polymerize. You should use a fresh APS solution (at room temperature) to optimize the polymerization.
The resolving gel takes 30-45 min for polymerization. However it should be kept in mind that 10% APS used should be freshly prepared and TEMED used should be properly stored in brown bottle (with minimum light exposure). After casting the resolving gel, butanol saturated water should be over layed so that the gel doesnt come in direct contact with the air as it may hinder in polymerization. Once polymerization is done, discard the butanol saturated water and cast with stacking gel which also takes around 30-45 min for polymerization.
It is correct. after 40 minutes, you can see that the gel polymerise, but this is not the end of the reaction. further polymerization within the gel takes about 12 hours and after this time there is a complete cross-linking
It depends on which gel it is. I prefer to leave polyacrilamide gels overnight before adding the stacking gel to allow complete polymerization. This gives me very good seperation of my proteins, in addition to other factors like current. For agarose gels, around 45 minutes is good enough to polymerize and run your DNA samples. Proteins are abit more sensitive than DNA so you need to be very keen if you are working with proteins and especially if you need your protein in its native form. All the best
If you use fresh reactives, the gels will polimerize in 30 minutes. The most important is fresh ammonium persulfate(APS). When I buy APS, I dissolve the complete reactive and store in 1.5 ml at -20 grades.
SDS-PAGE gels polymerise in 10-15min in BioRad minigel set up. We normally use few granule of APS salt instead of freshly made APS solution.
Hi Mohd!
For agarose gels, the polymerization takes around 45/50 minutes to run your DNA samples.
40 min at room temparature for 5-15% PAAG using TEMED-APS catalyst pair. Too quick polymerization leads to uneven porosity.
Yes, for polyacrylamide gels its best to cast the separating gel overnight, and cast the stacking gel next morning, then run after a couple of hours. In most equipments today you can cast multiple gels, so, while the first gel may be ready to use by noon the next day, the remaining gels are ready to be used at will. However, there have been times when we have needed protein profile information at very short notice, and then, we cast our gels the same way exactly as we do for the overnight gels, but pour the stacking gel about 20 minutes after the separating gel has been poured, in fact as soon as the gel interface becomes visible. Unless you are resolving proteins that are very close in molecular weight, this 20 min cast works well. But for higher resolution, I would prefer an overnight casting of the separating gel.
We used to cast several gels at once and store them in a wet box/wrapped in wet paper at 4°C for days up to two weeks. You will see, when they start to shrink, then discard.
For the original question: 0,5-1 hours per gel (separating/stacking) depending on the gel size. 30 minutes for an agarose gel, you might speed it up (15 minutes) when putting the agarose gel into the fridge.
For agarose gels it depends of the percentage, you can see the gel is ready when it turns blue or grisaceus, normally as it was indicated by Vivica Grotelueschen1,15 min
For SDS-PAGE I used to put a tip into the rest of the mixture and when it is hard, the separing gel is ready, you can use 1ml-0.1%SDS to accelerate the polymerization over the casted gel or butanol. For the stacking, I make the same thing with the rest of the mix. Separating gel takes aprox 30-45 and stacking 15 min. During this time, you can prepare your samples. Gels can be stored at 4ºC for the next day o for the followings three days. I have tried to use fresh gels, not older than one week. Good look
it took 10-mints time; you need to keep APS as good as possible ipolymerizationdepends completely in it
it takes exactly 40 min separetly for resolving and stacking gels of polyacrylamide and agarose 30min.
The glass container foe gel should be clean and after addition of APS please don't shake it . It takes 15'' for a PAG to polymerize.
Hello, people,
Are you still using stacking (conentrating) gels? It is not rock. To obtain narrow nice protein bands just do not overload the gells (wells) with protein and let your sample buffer molarity be 10 times less than the molarity of separating buffer. The two-gel system is boring to handle with ;-)
If you are doing polyacrylamide gel electrophoresis then for polymerization of separating gel 30-40 min are required and for stacking gel 5min are required.
For agarose gel electrophoresis for polymerization 15-20 min period is sufficient.
It depends on your surrounding temperature too because the gel polymerization time period for PAGE seems to be 25-30 mins for separating gel and 5 mins for stacking gel at Air-conditioned temperature (ie., 26 degree celsisus). Similarly for Agarose gel electrophoresis polymerization of gel occurred within 6 mins.
In both cases there was approximately an 4-6 mins delay in gel polymerization when the whole process was carried out at normal room temperature (ie., 37 degree Celsius).
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