EMSA Shifted band in Free probe only lane - (Mar/06/2014 )
I have been working on repeating an EMSA shift from the lit. I am using DIG gel shift kit vers 2 from Roche.
I have compl. synthetic ssDNA oligos 30bp. Anneal myself. then label while labeling a pre-annealed ctrl.
After running my gels on novex precast 6% DNA retard. gels I get a nonspecific band in my free probe only lane as well as my labeled probe with pure proteins but not in my controls. See image please.
What is this non-spec band how do I get rid of it?
Sorry for the late reply. Hope you have already solved the problem.
The gel patterns look very weird to me. since you also got the nonspecific shift (band) in your free probe lane, I doubt it is a shifted band. It appear to be a higher sized DNA band. Is It possible when you do annealing, the oligos not only formed duplex DNA, but also concatenated to give a longer dsDNA.