We are working on PAGE (Polyacrylamide Gel Electrophoresis) for resolving the amplified DNA samples of wheat crop. But when we tends to prepare PAGE gel, after its solidification, when we remove the rubber gasket the air bubbles fill the whole plate.
Anyone please tell us how can we prevent air bubbles in it?
Thanks.
It sounds like air is getting in between the glass plate and the solidified gel. Maybe this can be prevented if you keep the plates clamped together most of the way around as you take out the gasket. Or switch to pre-cast gels.
Possible reasons-
1. Change the casting gel apparatus. It may be some problem in the rubber gasket or something wrong in the apparatus, so try to cast the gel in new apparatus.
2. Pour the running gel, then add the butanol, polymerize it for 40-50 min and then rinse the butanol with distilled water.
3. Before adding Ammonium Persulphate (APS) and TEMED to the solution, thoroughly mix the solution, settle it down for 5-10 minutes and after then add the APS and TEMED and mix the solution very gently to avoid any air bubble. Carefully pour the gel solution between the plates (gel casting apparatus).
## PAU have excellent experimental facility. If the problem will still persist then you may contact to Agricultural Biotechnology Department, PAU.
All the best.
This picture looks like air between the gel and the plate, as I guessed. Working with very clean plates, as Asa Wu suggested, may help reduce this problem.
Did you degas the acryl/bis-acryl solution prior to adding APS and TEMED?
Dear Kunal,
Please degas your acrylamide/bis-acrylamide solutions either using sonicating water bath or vacuum. Degasing (removal of oxygen) helps with efficient and uniform polymerization of acrylamide. In my opinion, your problem is non-uniform polymerization. Just give degasing a try and let us know if it helps.
Dear Tao,
Currently, I am not facing this problem anymore. I have not change any component in preparation of gel, neither degas nor reducing the amount of APS or TEMED. I am using the same concentration as I used earlier.
I observed that PAGE glass plate compatibility (means local made glass plate and glass plate from manufacturer) may be the problem. Now, I am using the same set of glass plates. If I am using base plate manufactured from local market then the notch plate used also from the local market not from manufacturer.
Second thing is the gasket used to seal the ends of the PAGE plate should be adequately fixed or the ends of the plates may be double sealed using clear tape.
Third, apply adhesive or bind silane to the sides of the plate properly.
Hope this helps. Please share your observations also.
Kunal
Hello everyone!!
I've had same problem lately.
Do you think it affects running of the samples in gel?
Or should I make a new gel?
- Badges
- Science method