After I pouring resolving gel, I put a layer of isopropanol. Resolving gel looked polymerised well.
However, after I pouring stacking gel on top of it and letting it to be polymerised, I found that some of the stacking gel leaked.
How does it happen? How come there is a leaking on stacking gel when my resolving gel does not leak at all?
You still have to put into consideration the packing of the pack then allow the resolving get to polymerise to a reasonable extent, consider the time of polymerisation of the stacking gel, seal up loosed areas and pour carefully.
Thanks for the reply.. I will probably mark the "height" of the resolving gel before I pour the stacking gel. Therefore I would know if there is leaking for the resolving gel.
Today I prepare 4 gels. 2 of them were good however there is a leaking of stacking gel for another 2.
The stacking gel should be upto 1 cm below the wells.
I do not know about your unit, but after assembling your unit, you can pre check it by adding water and see whether it leaks. This will assure at least you have assembled properly the gel casting unit.
I agree to Sarang Sapre, Pre-checking your devices is important.
In our lab, we used to deal with BioRad system, and I found an interesting situation that is some certain combination of sheet glass and holder will leak. In my opinion, the sheet glass and silicon stripes somehow may be frayed somewhere.
In your case, I guess the frayed area may be on the upper part where next to the stacking gel. Only in guess.
hope it will be helpful.
Thanks for answer provided from both of you. I do appreciate it..
I guess my situation is more towards what Samuel mentioned as I do not see any leaking for my resolving gel (bottom part of the sheet glass).
In addition, I have a condition that there will be some air bubbles on my well, causing uneven shape/height/volume of the wells.
Do you know what is the reason and the solution of it?
In regards to the leak that you are experiencing, it can derive from the gaskets or from the glass (depending on your system). That can be checked with DDW.
In regards to the bubbles, you can either degas the gel before pouring it or simply put more gel (let it spill) and then insert the comb diagonally.
Good luck.
Thanks, Ariel.. regarding to the bubbles issue, my experience was I saw no bubbles after I inserting the combo. However, when the stacking gel polymerised, some of the wells formed 'lower' compared to other..
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