dimers of dna (not primers) following digest or pcr? - (Apr/25/2006 )
I can hardly see your fragment through all this smear. And the marker? It doesn`t show distinct bands at all which is really a bad sign. I mean maybe your staining solution is way to sensitive or contaminated with DNA or so and thats why you end up with this "dirty gels". Or you incubate it too long.
I see sth. like that only if I put too much EtBr in my gels. Basically everything is orange then.
I would try it with EtBr. It is the standard technique to stain gels. And it only takes a few seconds to add it to the agarose. And I would use new buffer to run the gels in. You never know whats swimming around in it.
Loading 50 µl in one well is ok and works fine. If you seperate your PCR mix into two wells, the signal of your fragment is weaker and you have to cut out more agarose which isn`t too good for purification.
Hello folks, I went through all your posters. But none of you seems to understand G.Star's question. It is not about the complicity of his/her PCR template, the way he/she stained the gel or any possible contamination left in the well. The double sized bands are real. They are the Holiday junctions formed by homologous recombination of the products from PCR or restriction digestion. They form because the double stranded DNA is in the relaxed conformation and has gone through denaturing and re-naturing steps, like what happens in PCR.
Please see the attached EM picture of the holiday junction and an example of gel showing the PCR products and the double sized bands on the agarose gel.