Very easy question. - (Jan/26/2006 )

Does uncut plasmid DNA migrate in a gel (1% agarose) differently to once-cut (linearized) plasmid?


Thanks.

[Am doing my first ever cloning. Sure hope my colleagues don't read this ]

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Hi-

It sure does. Uncut plasmid is usually "supercoiled," so it tends to run below its expected size on an agarose gel. If you nick it it should run right at the expected molecular weight.

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In fact, if the uncut plasmid is from a routine plasmid prep, you'll likely see several forms, all migrating differently.

This question comes up with some frequency -- see the thread here, for example...

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you can only estimate the lenght of your vector by the cut plasmid (linear) not the uncut one which can come out as two or three bands....all are different forms of supercoils....

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I belive only one band is supercoiled, the others are relieved of supercoiling.

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really ? i dont know....i got this explanation from the web....in the lab i have been told that they are some linear some supercoiled....but when i searched the web it seemed that you cant get the linear form unless you digest the vector....so all the forms you see will be supercoils.... maybe relaxed supercoils....

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check this -> http://hg.wustl.edu/hdk_lab_manual/plasmid/plsmid10.html

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Kersten's link is correct. If you want to see all fragments: just put A LOT of your prep on gel (a lot more than you would normally do).

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I was always taught that the different bands were due to varied degrees of supercoiling...someone must get to the bottom of this!

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I was always taught as is seen in Kersten's thread...

this one too:here

I tried briefly to find a reference, but I suspect it's way back in antiquity...I suppose you could design a very complicated experiment to determine the exact compostion of every band if you wanted to, but I suspect it's been done

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