extra band after gel extraction - (Dec/30/2005 )
i did gel extraction using QIAquick gel extraction kit. I've got my DNA fragment. However, i also got some unspecific bands (few smaller size than my expected band size and two larger size ..!). So, i heat up the purification product to 90 Celcius. After heating, i've got 2 bands: one is my DNA fragment and the other one is the unspecific band which is larger than the expected DNA fragment.
May i know what happen to my purified product(oh or perhaps i should say so called purified..) since i've done gel extraction and logically i should get only an expected band..? Any idea what's that larger band and how can i eliminate that extra band ?
ps. hmm, i've actually posted this under someone's topic, so i think i should post it again..heh.
The other band could be ssDNA although these usually run smaller
Are you extracting from a restriction digest? Is it possible that the DNA did not cut to completion? If the larger band is weaker than the smaller band that could be your problem.
i've faced the problem before. it was a problem of quantity.
Before extraction the DNA in the band of my interest was in too larger quantity for well separation of the close sized ones.
Try running the DNA on gel for longer period so that even if there is no complete digestion your band of interest is clearly separated. Carefully cut only the band u want to elute.