I am using Sequei-gen GT (BioRad) system with well forming comb for SSR analysis. i am facing a problem, the gel is not polymerized near the comb and well not formed properly. Near the comb the solution remained as it is and doesn't polymerize. Please suggest the possible solutions..
Hello Ravinder,
Does the problem occur only with non-denaturing gels ? Otherwise, poor polymerization could be due to:
- Ammonium persulfate solution too old or ammonium persulfate powder decomposed. Beware of old batches of persulfate that are caked. Try increasing the amount of persulfate added for polymerization
- Too much dissoved oxygen in the acrylamide solution. Try degassing before pouring the gel
- Polymerzation inhibitors released by the comb. I once had problems with Teflon combs. Try cleaning the comb with ethanol and dry it
- Waiting too long before inserting the comb. Pour the gel and insert the comb as rapidly as possible after adding persulfate + Temed and don't move it afterwards.
- Not enough acrylamide, or leaks. Try to fill mold as much as possible without spilling.
- Concentration of acrylamide/bisacrylamide not adequate. Check published recipe.
Good luck
Pierre
Hello Ravinder,
Does the problem occur only with non-denaturing gels ? Otherwise, poor polymerization could be due to:
- Ammonium persulfate solution too old or ammonium persulfate powder decomposed. Beware of old batches of persulfate that are caked. Try increasing the amount of persulfate added for polymerization
- Too much dissoved oxygen in the acrylamide solution. Try degassing before pouring the gel
- Polymerzation inhibitors released by the comb. I once had problems with Teflon combs. Try cleaning the comb with ethanol and dry it
- Waiting too long before inserting the comb. Pour the gel and insert the comb as rapidly as possible after adding persulfate + Temed and don't move it afterwards.
- Not enough acrylamide, or leaks. Try to fill mold as much as possible without spilling.
- Concentration of acrylamide/bisacrylamide not adequate. Check published recipe.
Good luck
Pierre
Please change either APS or TEMED. The polymerization occurs because of these two and act as electron donor and acceptor. If there is any problem in these the polymerization will not occur properly.
Yes, I agree with Pierre and I would also recommend preparing a fresh APS solution because I had encountered almost similar situation some time back.
We had also prepared a checklist of possible reasons like increasing the acrylamide/ biscrylamide concentration but when we prepared fresh APS solution it worked for us.
Have you recently changed the acrylamide/ Bisacrylamide? because there is a possibility that the new batch of Acrylamide/ Bisacrylamide might have some problem. If not what is the concentration of acrylamide/ biscrylamide in the stacking gel that you are keeping?
Because in normal practice a concentration of 4-5% is being used for stacking gel. please check what is your concentration.
hello Gaurav.
i am using 6% gel for the separation of SSR markers and i didnot changed the Acry:Bisacry.
If this really is restricted to the are near the comb, it cannot be the acrylamide stock, TEMED, APS or any other component, otherwise I think the whole gel would be affected. But a 6% gel may take some time to polymerise and areas in contact with air might be affected. I suggest you make sure the comb is completely immersed in gel mix. Monitor the excess mix as an indication of when polymerisation is complete. Then rinse out the wells with running buffer before loading the samples.
When I have had this problem it has always been the APS (even though freshly prepared), usually because the solid absorbed moisture (and maybe degraded as a result?). I now keep the solid APS in a dessicator and have had no further issues for decades (but still change it every few years). Another thing, though, is that air inhibits polymerization, so you either have to "sink" the comb into the stacking gel, leaving no air gaps (which is what I do), or else overlay the exposed gel with water-saturated s-butanol or the like.
Norbert, et al.: I used to think like you, but then a new post doc showed us that 10% APS in water in frozen aliquots works perfectly. So now I have a box of small aliquots (200uL) in the minus 20 freezer that I make up only occasionally: saves me a lot of time!!
I've done that too, but I'm never sure how long to trust them, especially from a -20 freezer that is heavily used. To alleviate that stress, what I do instead is tare a glass dispo tube (less issues with static if you use glass), toss in a small scoop of APS (which from years of experience I know is about 50 mg), weigh it to 4 decimal places, and pipet in 10 x the weight in dH2O (e.g. 0.0481 g would require 481 µl). Vortex and it's ready to use. I know it's not precisely 10%, but it always works. It takes me about a minute, casually. Less time than walking to the freezer, rooting around for a box of tubes, and thawing an aliquot (but I am lucky in that I have a good balance next to my bench and everything I need, including the dessicator with APS, right on my bench... which may be limiting for others) : )
Hello Belen,
Mainly, oxygen cause this problem. that may be dissolved in solution or contact near comb area.
Use long teeth combs wiped with distilled water,
Proper degas the solution before injection between the plates.
Good luck.
My dear friend: Do thanks to all your well wishers who have poured in their hearts to help you. I too have faced such problems earlier. Just note that the brand of acrylamide often is the culprit. Change the brand and see. Secondly clean the comb very thoroughly or still better use a new comb and you may be surprised to see that the problem disappear.
Oxygen is a radical scavenger and therefore prevents polymerisation. Carefully degas your gel mixture (vacuum or sonicating water bath) and when you mi in TEMED be gentle not to dissolve air oxygen again.
With some manufacturers you get air in between the gel and the comb, which causes ill-formed wells. In such caeses have your craftshop make better combs (for details, see doi:10.1016/S0003-2697(02)00639-5).
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