If you are sure that everything else is the same then it sounds like it was your Acrylamide:Bis that was the problem. I would double check your Acrylamide:Bis to make sure you have the same ratio of acrylamide:bis that you have been using in the past. There are usually three ratios 37.5:1, 29:1, and 19:1 and the different ratio will definitely affect your polymerizing time. I have read that the optimal polymerization time for overlaid gels is when the visible polymerization takes place in 15-20 min; and the optimal polymerization time for nonoverlaid gels is when visible polymerization takes place in 8-10 min. You can control this by varying the amount of TEMED and APS added. I hope that this helps you out.

Kyle
Laboratory Technician at Protea Biosciences
www.proteabio.com

can you comment on what happens if there is an incorrect amount of APS and TEMED?

would you observe the differential polymerization or even the leak at the bottom of my gel?

There are many reasons why the right amount of APS and TEMED are important. The reason why it is important to get visible polymerization in between the times I listed above is because if you don't then oxygen will begin to enter into your acrylamide and inhibit polymerization. You may have noticed this when you think your gel has polymerized and you pull out the comb and the wells have not fully polymerized or did not polymerize at all. Also when you reduce the amount of APS and TEMED it increases the length of the polymer chains, increases elasticity of your gel, and lowers the cloudiness of your gel. Which are all good things. However, if the TEMED and APS are too low then the gel does not polymerize fast enough and the oxygen begins to affect your acrylamide. The most common evidence that there is too much TEMED or APS, is the schlieren pattern. The schlieren pattern is the swirls that can be seen after polymerization. The schlieren pattern can also be caused by the poor mixing of the solution after adding the APS and TEMED. To mix properly swirl the solution approximately ten times. The swirling motion reduces the chance of inducing oxygen into your gel solution. It sounds like you are on the right track for the amount of TEMED you use. The amount of APS you use sounds a little high, but I could be wrong. There are so many things that can affect the how much you use. I think Bio-Rad recommends you start use around 50ul of APS per 10mL of gel solution. Protea on the other hand recommends you to start with 75ul of APS per 10mL. This is because we have a special recipe for our gels that give them a drastic increase in strength over the average gel. So it depends on your recipe but those are some starting places.

The leaking of your gels could be caused by the Bio-Rad apparatus that you are using. Make sure the sponge seal at the bottom is clean. If the gel solution is not cleaned off after every use then the gel solution can gunk up the sponge and you can lose a good seal.

I hope this helps you out. If you want to take a look at Protea's gel solutions and precast gels click these links.

Protea’s Ready to Run Gel Solutions

Protea’s Precast Gels

Good luck,

Kyle
Laboratory Technician at Protea Biosciences
www.proteabio.com

Hi Kyle,

thanks again for all the help!

i recently attempted the gel again and was much more patient and allowed the gel to sit for 30-60min. I checked it at 30 and it seemed solidified but the excess in the tube was not fully polymerized so I allowed it to sit until 60 minutes.

When I added the stacking gel I noticed that five minutes after filling it to the brim, the walls of the ends would leak down to half the wall height. Is this either too slow polymerization or too much of a leak through the sides of the glass plates?

I checked the bottom of the glass after both running and stacking gels were solidified and noticed there was still a 1mm space at the bottom but interestingly enough the space formed a concave with the apex 1mm up towards the center. I am assuming improvement in the seal?

Regardless of these observations I cannot help thinking it is the acrylamide:bis stock that has loss its original concentration, and thus slowing down the polymerization? Is there anyway to test for this to verify my suspicion?

Thanks

Hey Azrael201, sorry about the delayed response.

When I have used the Bio-Rad apparatus with the glass plates, I have also noticed frequent drops in the outside well walls. It seems pretty typical and it should not be a big problem unless the sample you are loading is at such a volume that it flows over smaller wall. If it is important you could try a little bit more TEMED to shorten the polymerization time.

As far at the 1mm space at the bottom of the gel, that is probably due to the sponge seal coming up in between the glass plates. I have also seen this before using that Bio-Rad system. This also should not be a big deal. It just means that you can’t run your gel that extra millimeter.

If it is important to get rid of the leaking wells and the 1 mm space at the bottom of the gel then I recommend you looking into precast gels for your apparatus. Bio-Rad makes precast gels for their gel box, however so does Protea and Protea’s cost less. Protea also sells the empty plastic cassettes; these will not leak and do not need a special gel pouring apparatus. I know Precast gels aren't practical for most labs but I throw them out there because they are extremely nice to use and make the gel process much easier. Also when trying to produce a gel for publication, these gels go a long way in producing quality images.

The Bis/Acrylamide stock solutions can go bad, but that is usually after about a year, as long as it is stored at 4°C. It is important that the Bis/Acrylamide solution is stored at 4°C, however once it is added to make the gel solution it becomes a lot more stable at room temperature.

Kyle
Laboratory Technician at Protea Biosciences
www.proteabio.com