Please help with high-molecular-weight DNA gel electrophoresis! - (Jan/25/2006 )
I'm trying to separate genomic DNA on a 0.5%agarose in 1xTBE.
I don't have a PFGE equipment for that, so I'm using standard agarose gel electrophoresis.
I know that 50kb DNA can be run on 0.5% agarose on 10v for 24 hrs at 25C. But how should I control Temperature, EtBR,etc? Should I just check every couple hours, and add buffer, EtBr? My previous experiments with that failed.
Temperature shouldn't be too much of a problem. I don't think (not positive though) that at 10v would make the TBE would heat up enough to be problematic. If you are concerned about EtBr diffusing out of your gel, you have the option of staining it after it is done running. This is slower than adding the EtBr straight to the melted agarose but you won't have to worry about it leaving your gel.
You can also add EtBr to the running buffer at the positive end of the gel box.