Excess acrylamide in wells - (Apr/20/2009 )

you can do that but water can diffuse into and mix with and dilute the stacking gel buffer.

with electrode buffer you are putting the gel into the same condition in which you will run it.

Hi
I have had the same issue with Biorad Mini Protean while using a 1.0mm spacer and combs combination.

However, I observed that the problem was not so great when I removed the combs soon after the stacking gel polymerized (like within 10min).

We did call Biorad who said they have heard the same prob before, and sent us replacement plates and combs, but the problem persisted when I left the gels+comb for a longer duration (>20-30min)

Hope this helps.


Cheers

Rob Steuart on Apr 20 2009, 09:57 PM said:

Well, APS-TEMED polymerization takes place in the absence of oxygen.

First: Try adding an amount of gel between the plates so that no extra-gel flows out of the gel when you insert the combs. As this outside gel will be exposed to air and take longer or wont polymerize. Also try increasing APS to 100ul 10% and TEMED 10ul per 10ml of gel. APS activity will fall to half of its activity after a litle time in the fridge or frozen.

Second and highly recommended for everyone: add riboflavin-5-phospate to the stacking gel. Rifoflavin will polymerize in the presence of air, so comb or plates alteration wont matter much, so that you'll have nice formed wells.

Feelcontraire on Nov 18 2009, 12:25 PM said:

adding more aps-temed will create a gel with shorter strands and will be more fragile.

polymerization with riboflavin (which requires light activation) will be effected by oxygen. it is still a free radical reaction. we use it to polymerize the stacking gel of our non-denaturing page.

mdfenko on Nov 18 2009, 01:17 PM said:

Yes, adding more TEMED will make the gel more fragile but as long as it is kept in the 11-1ul/10ml it's ok. Notice it was an advice for people having trouble with there well formation. TEMED also oxidises by time and people sometimes use lower ph gels which reduces TEMED activity for this reason when polymerization doesn't work properly I recommend pointing higher on APS-TEMED values.
If gel fragility is a problem 10% glicerine can be added which gives nice flexibility.

In fact litle oxygen is enough for riboflavin polymerization, but strong degassing may inhibit the polymerization.
You can also use it in denaturing gels, and I strongly recommend it for people that need nicer wells. Also riboflavin is a mild oxidizing agent so it wont add much extra-free radical formation that may interact with proteins in the gel. Riboflavin is added to the standard plus the standard APS-TEMED.

Do you think i would put a question up like this if i hadn't tried evrything i can think of.



I hope that bad karma come back to at some point




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Hi Aris, What u used? Can lend to me as well? Because what i wonder for me is the difficulty to wash the acrylamide in the well.
Question for gbbg, hi, I m also using Biorad Mini Protean (1.0 mm), I having the exactly same problem as u, i also found that it is easy for me to wash if i removed the combs quickly after stacking gel polymerized. i using 10 well comb, theoretically, 44 ul volume of sample can be loaded, but i only able to load 20-25 ul. Should i ask the biorad ppl change for me also? but u say the problem still persisted aft u changed.

Hello. I had the same problem, actually am still having it in every gel. I am sure I use the correct size comb.
The solution I could find is, taking a clean gel loading pipette tip (the ones with extremely thin and long tips), disrupt the gel in the well and try to remove it by kind of stirring inside the well creating a vortex with the pipette tip again.
Works fine with me now, although might take 20-30 minutes to complete, so I suggest you be patient.
Good luck to everyone.

in fact my problem is not excess acrylamide in well but is refered to wells....
I'm having exactly the same problem as u JEE (Biorad Mini Protean (1.0 mm). i'm using 10 well comb, in theory 44 ul volume of sample can be loaded, but i'm only able to load 20-25 ul. If i need to load more than 25 ul, can i run a few min and reload the sample ?

tiki on Fri Oct 29 15:58:40 2010 said:

you may be mixing your sample with the electrode buffer when you load the gel. does your loading buffer have sufficient glycerol or sucrose (or something else) to ensure that the sample is significantly more dense than the buffer? are you loading too fast?