Hi all,
I have tried many method such as parafilm, 5% agarose. My gel is still leaking. I have read the top comment on the youtube video (https://www.youtube.com/watch?v=b45nSOyPP_4). He suggested to line down the gel setting stand horizontally. Has anyone tried this method yet?
But when I finish pouring resolving gel, I need to add water on the top to prevent oxygen get in. If I line it horizontally, I am not sure if the water would stay at the top part of the gel due to the gravity.
Thank you
https://www.youtube.com/watch?v=b45nSOyPP_4
Hey,
When setting up the instruments, you have to confirm that:
1. The bottom edges of the glass slides are in the same position.
2. After setting the slides into the plastic frame, it should be adjusted accurately with the large frame.
Usually these are the main things to set up. To be more sure you can put a pipette tips or a pen on the glass clip to provide more pressure on the glass, so that there will be less chance of space to leak.
Additionally it also depends on the % of gel you are making and how well you mixed TMED.
And after pouring the resolving gel you can use isopropyl alcohol (IPA) instead of water. IPA will not pass through the gel. after polymerization you should remove IPA and wash with 1ml of water. And then as usual........
Hope it will work and best of luck.
Hey,
When setting up the instruments, you have to confirm that:
1. The bottom edges of the glass slides are in the same position.
2. After setting the slides into the plastic frame, it should be adjusted accurately with the large frame.
Usually these are the main things to set up. To be more sure you can put a pipette tips or a pen on the glass clip to provide more pressure on the glass, so that there will be less chance of space to leak.
Additionally it also depends on the % of gel you are making and how well you mixed TMED.
And after pouring the resolving gel you can use isopropyl alcohol (IPA) instead of water. IPA will not pass through the gel. after polymerization you should remove IPA and wash with 1ml of water. And then as usual........
Hope it will work and best of luck.
It seems like you should replace the grey seal in the bottom of the casting apparatus. Before getting new ones, try to flip them upside-down, perhaps the bottom side is less worn out.
It is also recommended to check for leakage with water before pouring the resolving gel mixture.
Good luck!
One way is to use vacuum fat (fat for desiccator) put small amount on the bottom angles of the apparatuses.
However, I succeed with SDS-PAGE gels.
Good luck.
I have found that rolled up paper towel wedged in the clamp increases the pressure on the glass and reduces leaks, and also check that there are no chips in the glass plates.
Make sure the grey seals used on the bottom get washed thoroughly too because the gel builds up in them, making them hard and more likely to leak.
Hopefully you don't have any more leaks!
Leakage happens usually because seals loose flexibility or because of their mechanical damage (burrs, micro-cracks etc.). If you cannot exchange seals, you may use some tricks (from my experience):
Carefully wash all parts of sandwich with defatting solutions, especially glasses, and dry them carefully before assembling. I used isopropyl alcohol at the last stage.
Make hermetization of sandwich bottom perimeter with melted hot agarose (I use 1.5 %). Try to put into all splits. Its fluidity depends on the temperature, so use it hot. Try to apply it on the side parts of the sandwich.
It is possible to calculate approximately volume of leaked solution and to flood more resolving gel mixture into the sandwich. Leakage stops when polymerization occurs. If your leakage is not very intensive this may help. Additionally you can wait for several minutes before flooding of prepared gel into the sandwich. This will make shorter the period before polymerization. But you need experience otherwise your gel will polymerize in glass.
Just as Yuri stated emphatically, i clean the glass plates and spacers with 70% ethanol. After assembling all parts and fixing them pipette about 1 ml of 1% hot melted agarose and dispense via one side of the assembled plate and spacer. Then, tilt the set up so that the agarose lines at the bottom uniformly. Leave for about 5 minutes and it will solidify and then you can add your gel mixture accordingly.
This method works well even if there are cracks at the edges of your plates. Just ensure that the cracked edges face up while the smoother edges face down.
All the best.
i usually push the bottom of the gel casting plate about 1-2 mm out of the bottom of the holder so that when i clamp it into the stand the plate will be pushed into the foam section at the bottom more than it would be if the plate was simply level with the holder. so its less likely to leak.
I have always done vertical casting. In fact, vertical casting done on a leveled,smooth surface ensures that the plates are lined properly. Also apply some extra pressure on the holder clips. The caster apparatus usually uses some kind of gasket that prevents leakage, make sure you place your gasket properly. Last, as a desperate last attempt, when the gel is leaking from the bottom immediately add more of TEMED and APS directly onto the leaking mess! it will itself form a plug sooner than the gel will take to solidify, thus restricting further leakage.
Before pouring in the acrylamide mix, pour in water to check for leaks. This prevents the neurotoxicity unpolymerised acrylamide from needing to be cleaned up. Tip out the water after each test for leaks. To prevent leaking, you can add two layers of filter paper under the rubber seal at the bottom to add extra pressure. Ensure that the glass plates are level at the bottom.
I have tried some ways that really work excellent. similar with Abhishek Saxena, you can firstly prepare 1ml gel with a high concentration of TMEND. Then, quickly add to the gasket forming, after that, put the plate immediately onto it before solidifying. The solid gel can form a reliable barrier, so that you won't worry about the leakage anymore:)
Make sure that the glass plates should not be broken or chipped from sides and leveled equally during the clamp. I always use rubber gasket below the gel plates. If you also use rubber gasket then make sure that it should be tight enough to prevent leakage. Thank you.
at first just seal bottom with normal tape. just cover it. much wider tape much better. now use. use 2 clips then cover bottom with tape. then send it cassate. now as usual. i can say it will protect 100%. paraflm is also good in same way.
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