Even if you do not have visible airbubbles when you cover your separating gel with isopropanol (or water-saturated n-butanol), you can still have air dissolved in your separating gel mix. This happens when you mix too vigurously. When you pipet the Water, Acryl/Bis, Tris, SDS, APS and TEMED, you should swirl very gently and slowly after adding TEMED, don't shake the mix, just a gentle rotating motion, as if you were appreciating a good VSOP cognac in a glass, and then pour it slowly between the assembled plates. When all the reagents are good and work for other people in the lab, whenever a student has problems with uneven polymerisation I ask them to show me how they mix the reagents, and I find that invariantly they have mixed too brutally, dissolving air in the process. I also strongly recommend Andrés' advice, make 10% APS aliquots and store them at 20ºC.

Hi Murugadas A

Adding isopropanol to the top of the separating gel will definitely give you a nice level gel, and as far as I am aware it is a very common practice. Once you have filled your separating gel mix to the right level, then just add half a mL or so on top. You will see straight away that the separating gel levels off and the isopropanol will just float on top. Once the gel has set, rinse out the isopropanol with water and continue with the stacking gel.

Jacob

Hi,

If your problem is air bubbles, isopropanol should do the trick. What you may have noticed is that you often get air bubbles or froth in the gel mixture when you mix it. This is due mainly to the SDS; all detergents have this frothing property. Once you pour this mixture into the gel cast, it can be difficult to prevent the bubbles from pouring into the cast too. Adding isopropanol (~ 150µL per 5mL gel) immediately after you pour the resolving gel will eliminate this problem and leave the top of the resolving gel nice and flat. Leave the gel to set (~5mins for a 5mL 10% gel; lower percentage gels take longer to set. I usually prepare ~1mL of gel mixture extra and leave it in the tube; once this has set, your gel should have set too, and you can check this by simply turning the gel slightly sideways like turning the steering wheel of a car; the isopropanol should move to the side you are turning the gel but the solid gel will not move).

When waiting for the resolving gel to set, prepare your stacking gel but do not add TEMED just yet. Once the resolving gel has set, you want to briefly and gently wash the top of the gel with distilled water to remove all traces of the isopropanol. Turn the gel upside down and use the corner of a tissue to dry as much of the water as possible. Now you can add TEMED to your stacking gel mixture and pour it on top of your resolving gel. Add your combs and you're good to go!

However, if your problem is indeed uneven polymerisation of the gel, I recommend thoroughly mixing the gel mixture after adding TEMED; invert the tube ~10 times. This mixing will introduce lots of air bubbles but these bubbles can be eliminated using the method described above. For three 6mL (5mL resolving + 1mL stacking) 10% gels, these are the mixtures I would use:

Resolving gel

Water: 5.9mL

30% Acryl/Bis solution: 5mL

1.5M Tris: 3.8mL

10% SDS: 150µL

10% APS: 150µL

TEMED: 15µL

Stacking gel

Water: 2.1mL

30% Acryl/Bis solution: 500µL

1.0M Tris: 380µL

10% SDS: 30µL

10% APS: 30µL

TEMED: 3µL

Sorry for the long answer but I hope this helps!

Bilge

Totally agree with Jacob. I always use isopropanol and gel goes OK. Old APS is a frequent cause of no polymerisation. Make stocks of 10% APS at -20ºC, aqueous solutions of ammonium persulfate are unstable at room temperature.

Hi,

possibly your problem is in the Temed? I am sure you have all the knowledge and reagents right, so it is not a problem of mixing well before loading the separating solution. I always cover the surface of separating mix loaded in the wells with a layer, when I am in a hurry just water or water with SDS 0.1%. We did hundreds of gels in the old days with different % of C/T ratios, horizontal ones, IEF gels with photopolymerisation... Today, we use premade solutions, such as BioRad Criterion Stain Free gel mixes (separating with stacking together) ready to use. Usually it is good to cast two gels, so you may test one before running some important sample. Wish you will solve the problem!

Hi! Using isoprpanol won't effect in the composition of the gel..we sometimes use ethanol, butanol etc. to make the upper layer of resolving or seprating gel at same level..you should mix the gel nicely before pouring it in the casting assembly..try to make fresh 10% APS and add more TEMED..

Even if you do not have visible airbubbles when you cover your separating gel with isopropanol (or water-saturated n-butanol), you can still have air dissolved in your separating gel mix. This happens when you mix too vigurously. When you pipet the Water, Acryl/Bis, Tris, SDS, APS and TEMED, you should swirl very gently and slowly after adding TEMED, don't shake the mix, just a gentle rotating motion, as if you were appreciating a good VSOP cognac in a glass, and then pour it slowly between the assembled plates. When all the reagents are good and work for other people in the lab, whenever a student has problems with uneven polymerisation I ask them to show me how they mix the reagents, and I find that invariantly they have mixed too brutally, dissolving air in the process. I also strongly recommend Andrés' advice, make 10% APS aliquots and store them at 20ºC.

Sirs, Jacob Goodwin, Andrés González Rodríguez,Gaurav Chhetri I will try with isopropanol today

Sirs, Bilge Argunhan, Palmiro Poltronieri i don't think APS or TEMED could be the problem becoz every time i used to prepare fresh and the chemicals also purchased recently.

Sir, Jürgen Denecke, I ll agree with you next time when i do i follow all the points what you have suggested.

Thanks

As others pointed out, isopropanol will not affect the gel composition. You can add isopropanol right after loaded separating gel solution. Also, you have to mix the solution gently to avoid bubble. You may prepare your gel one day before use and keep at 4'C. Well, sometimes APS will also affect the polymerisation, especially when someone did not cap the APS bottle properly. I faced this problem a few times and after I changed a new bottle, then the problem solved.