Hello Friends,
Our lab is facing the problem of visualization of only single band of plasmid in gel instead of multiple bands. While the concentration of plasmid is much enough (around in micro gram quantity per micro Lt.). We are observing this difficulty by manual alkaline lysis method isolation of plasmid from E.coli
We are trying to find out the problem but we are unable to get any information.
Can any one explain that why it is happening and what are the possible solutions to get multiple bands of isolated plasmid?
Good day sir:
You can visualize a single plasmid in electrophoresis gel in three different conformations, hence 3 different bands for a single plasmid, you can guess wich of the conformation is the band according to the relative size between bands:
the first band (top-down) you will see, happens when the circular plasmid gets nicked at a single point and forms a linear conformation, much more "heavy" to pass for the agarose pore.
second band is a non supercoiled circular plasmid, hence it will travel relatively faster than a linear plasmid.
third band is the optimum supercoiled circular plasmid, since its supercoiled, its virtual space is much more smaller than the 2 conformations fore mentioned and travels till the bottom of the gel.
so:
how long are you running your electrophoresis? are you loosing bands?
wich one is the band you are seeing? it correlates to a linear plasmid? if not, you shouldnt worry, if you see supercoiled or circular band means that you are not degrading your plasmid and its a good result.
Hi Amber...!!
I think the single band in your plasmid preparation indicates presence of either of only one form of super coiled/ open circular/ circular plasmid. Hence the band of plasmid may not show the expected size. The other profiles of plasmid may also present but are in very low concentration which are not detectable on gel. But you can confirm it by doing restriction digestion either single or double digestion. If it gets digested with expected band size then it confirms presence of single form plasmid in your preparation.
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