Hello,
I have problem with loading wells in polyacrylamide gel. It looks like the gel around loading wells polimerize poorly. There is a lot of very thin layer gel in the wells after removing the comb. I have tried to flush it with the buffer, but the effect is way from desired. Any tips?
Dear Rafał Świechowski,
Mix all the ingredients of the loading gel properly and give more time to polymerize. Lastly very carefully remove the comb.
I had encountered the same problem when I casted my first gel. In my case I have two problems. First I thought that it will polymerize immediately, so I quickly poured the loading gel without mixing it properly. Second, allowed less time to polymerize. Believe me it need enough time to polymerize the loading gel than that of resolving gel. So do not do in hurry.
Hope these will help.
Shahnaij
I don't know if you do, but don't prepare the mixes in cold, and try to isolate all contact with oxygen when the gel is polymerizing.
Rafał Świechowski,
Place the gel in the electrophoresis tank; fill in the running buffer and then remove the comb so that you can avoid the damage that would possibly occur.
Always use 1X buffer..!
For quick polymerization check for the suitable concentration of TEMED..!
Hope this is useful..!!!
I hope adding a bit higher TEMED will help. Also, pour the gel-mix only after properly mixing the contents.
As for the thin layer of gel in the wells - be sure to use the comb that matches your gel frame in width. I know it sounds like a pretty basic and dumb piece of advice, but since (at least in MiniProtean system) the combs look exactly the same, it's really easy to use the wrong one, unless you label them by yourself.
Hello Rafal,
As I have faced same problem, I would like to suggest you the following:
1. Prepare your gel in a bit higher amount as required by you and let it polymerize in a small tube so that you can track your gel in the casting assembly. Wait till the gel has solidified properly in your small tube. If it doesn't solidify properly, there might be an issue with your chemicals
2. Make sure you are using APS and TEMED in optimum concentration
3. Check that you are not pouring too much of the gel, which may form clogs and result into poor quality wells
3. Be very careful while removing the comb. Try to lift it up slowly and parallel to the casting assembly.
All the best!
- Bhuwan
Hello Rafal
1-You should increase for both "resolving and Stack" APS and TEMED concentration just like 2ul
2- Mix it very well then pour it
3- After pouring the resolving .. You should incubate it at temperature 37 for 20 minutes Then pour the stack and also incubate at 37 until polymeriz .
4 - Remove your Combs after adding running buffer
All the best
Sally
Hi Rafal,
Give enough time for the gel to polymerize. Once you remove the comb Flush the wells with distilled water.
Gel pieces in the loading well can be removed using a needle.
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