I was working on vertical electrophoresis using polyacrylamide gel to separate DNA fragments, but I have encountered different problems, so I need your technical advice to resolve my problems.
The first problem is that the polyacrylamide solution takes a very long time to polymerize or solidify in the plate. I used 50ml polyacrylamide solution per plate by mixing 13.3ml (30% acrylamide bisacrylamide), 10ml (10x TBE), 0.350ml (10% ASP), 26.35ml (water) and 5µl (TEMED).
The second problem is that migrating DNA form a parabola shape after moving halfway from the well. So it ultimately gives the band of different sizes (those expected to be the same size).
The third problem is that the obtained band was not bold enough for scoring. I used ethidium bromide for staining the gel after electrophoresis.
To answer your questions:
To increase the polymerization time of your gel:
1. make sure your APS stock (crystals) is dry and make fresh APS. If the crystals are wet then throw the stock away and get new. This is usually the problem when gels do not polymerize in a reasonable amount of time.
2. Add more TEMED.
3. Put a flame (Bunsen burner) near the gel. Heat will increase the rate of polymerization.
Migrating DNA:
1. Be sure to load all lanes of the gel with at least loading dye such that the current/resistance is the same across the gel.
Gel staining:
1. Acrylamide gels stain very quickly with EtBr since they are so thin. If the gel is bright pink after staining, you may need to destain the gel so you can visualize the DNA. You should be able to see a few nanograms of DNA.
for proper polymerisation use fresh APS (when you dissolve it you should hear a "crisp" sound meanig the powder is dry)
let the mix (acryl+buffer but without APS and temed) warm up at room T° (after pooring your gel ytou can put a lamp (not a Bunsen burner!!!!) close to the plate to warm it up
the form of the band are probably because the gel warm up during running; use 1x TBE buffer and let the gel run slowly...
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