Why do I get two bands on the gel of a digested PCR product after purification w - (Apr/27/2018 )
I realize this answer is too late to help, but for any other readers, I would have to say that gel pictures would be of use in diagnosing the problem. It is possible you had two close bands (a "doublet") all along, but you could not see it because the mass of DNA on the gel was so great it made the two bands appear as one. When you only ran a little of the purified product, it was dilute enough to show as two separate bands. The digest could have been incomplete, leaving one band a bit longer due to still having the undigested termini intact. I assume you are using a high % agarose or using a special agarose for separating small fragments. But I'm guessing- pictures are worth a thousand words!