gel elution problem - (Aug/21/2007 )
Occasionally when I elute a specific piece of linear DNA from an agarose gel, either using commercial kits or a simple do it yourself protocol, the resulting sample contains not only the size fragment I cut out but a smaller and somewhat fuzzier fragment. This lower band, which is clearly derived from the larger, can be up to 50% of the material and is around half the MW of the larger. I was told once that acidic pH (samples were eluted into water with NaAc, so there would be acidic conditions) may be causing denaturation? although I'm unclear as to how effective EtBr would be in detecting this.
Anyone out there see something similar? anyone tempted to foward and explanation?
It has happened to me sometimes, but it’s just 20 % of the DNA. I think it could be denaturing DNA or your DNA was not well separated from other band, I don’t know….
Why don’t you elute in tris buffer or TE or water (being sure pH is 7) and compare???