I have a parental plasmid at 6.3kb, and this contamination just started appearing at 3.2kb. Everytime I run the gel out longer so I can separate bands and excise, gel purify, and retransform to purify my parental plasmid, the contamination disappears. I've tried variations in agarose %, time, and voltage. The variable that affects the appearance of the contamination is time, when I run it for awhile to separate bands it disappears. I ran the gel for a short time and carefully excised the band and ran a gel purification, but my spec reading was almost 0. What contamination could be time dependent, but continue to show up after quick change mutagenesis, minipreps, maxipreps, and many re-transformations?