Issues with making on SDS Page gel-Can not figure this out help!! :( :(

[manchestermicro]

Hiya,

Of all the issues I could have in the world my one is making an SDS page gel...

The stacking and resolving solutions re already premade for us...

However the issue I have been having lately is my gel (just the stacking not setting)

I basically let the stacking sit for an hour but sometimes its not done or the wells are very fragile and mess up very easliy..

I have made these before with no issues: here is the recipe I cannot figure it out:


Resolving 12%:

For 2 gels (made at the same time)
12% 14ml (7ml for each)
10% APS (100ul) (have checked this many times- used the same as I have before everyone elses gels turn out fine)
20ul of TEMED

Some ethanol to clear the bubbles rinsed out with water

Stacking 4%:
For 2 gels (made at the same time)
4% 4 mls (2 ml of each)
10% APS 50 ul
10ul of TEmed

Any help would be great thanks!

[protolder]

Hola, if before it works, now it has to be any mistake in the acrilamide% of your stacking gel mix. Although the normal % of acrilamide in stacking gels is 5%, check any recipe in internet, in this page for example in protocols. The amount of APS or TMED affect at the time of polimerization.Sometimes you have to change the 10% APS specially if it isn´t for PAGE. I always use for 5 ml of stacking mix 50ul of 10% APS and 5ul of TEMED.Try to add 1-2% acrilamide/bisacrilamide mix (30/0.8) to your current mix and your problem will be resolved(I hope). Buena suerte

[proteaHeather]

Hello Manchestermicro,
One of the major causes of polyacrylamide gels not polymerizing is due to outdated or bad APS and TEMED. I would try to make fresh APS solution first and see if that helps. If your TEMED has a yellow tint to it, it has gone bad and needs to be replaced.

The second thing you could try is to add slighly more TEMED to the reaction.

Hope this helps.

[manchestermicro]

Thank you all for your suggestions and help!! The TEMED we all use has gone off It happened to another girl across from me her stacking gel didnt polymerize and it looked like the four I had prepared that went wrong ...Thanks for everything!!