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plasmid DNA lab question - (Oct/16/2009 )

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Great, I can now not only answer his question but explain it to. Thanks again for your help.


Glad to help, relamberth. You had all the information and understanding, you just needed a little push -- exactly what we do here...

Not to confuse the issue, but you should understand that there are also other forms of plasmid DNA possible -- linear (which can in fact happen without restriction digestion -- what form would the plasmid DNA be in if it by chance it acquired two single-strand breaks on opposite strands but at the same location?), denatured (usually encountered when the DNA is left under alkaline conditions for too long), and various multimeric forms. These forms will also migrate through a gel at different rates -- sometimes running a gel of plasmid DNA (especially if it's improperly prepared) produces more than two bands.

Assuming, however, that your professor prepared the plasmid DNA for your lab with some care, which is probably a safe assumption, the two bands you saw were most likely the supercoiled and nicked forms -- they are the two most common forms seen when running out an untreated sample of plasmid DNA.

The take-home message is that the rate at which plasmid DNA migrates through a gel under set electrophoresis conditions (the voltage, gel concentration, buffer system, pH, etc.) is determined by the plasmid's size (mass) and its three-dimensional conformation. In a preparation containing just a single plasmid, the mass of these plasmids is all the same, so if multiple bands are seen, it indicates multiple forms of the plasmid exist in the preparation.

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