EMSA probe: two bands in free probe lane - (Sep/05/2007 )
I just ran an EMSA rxn using the Pierce lightshift kit and the lane with just my free probe has two bands. The lower (faster) band is much more intense than the upper band. I spoke with Pierce and they said that the lower band is single stranded and the upper band is double stranded. Is this right? If so, it suggests that my annealing didn't work well. I annealed using 1 pmol/ul of each oligo. I added each oligo to a tube containing enough STE (50mM NaCl) to get 1pmol/ul, heated to 95C in a heat block and removed the block from the heat and let cool to room temperature. I then placed the probed on ice, aliquoted and froze at -20C. Pierce suggested that I mix the two oligos together before diluting with STE and annealing, but I don't see why this would make any difference. Does this make sense? Should I anneal at a higher concentration? Any other suggestions?
Are you very, very, very confident of your DNA concentrations? If the labelled primer is in excess, then clearly you'll get the single-stranded form. How do you do the labelling?