I have been trying to clone a 1200 bp insert to PENTR vector.
After transformation and plasmid extraction, I run the super coiled and linearized vectors on the gel.
The length of linearized vector on the gel was correct (4700bp: is expected), but the length of super coiled was 8000bp (more than linearized form).
What is happened?
I know that super coiled DNA migrate faster in agarose gel but in my testing the supercoiled plasmid migrated slower than linearized form.
Assuming you run your gels without ethidium bromide, it is possible that your supercoiled plasmid is a dimer rather than the expected monomer. Does it run normally after linearization? Does it have the expected restriction digest pattern?
Hi Mo
to accurately size fragments based on size alone they must be linearised. This is because without linearization DNA can run in different conformers based on size and effective surface area (Stokes ratio) that incurs a drag factor and retards DNA
Yes you are right: Un cut or fully closed covalently circularised DNA, i.e. the traditional text book vector diagram, when in this nascent form, moves more quickly through a gel than the same plasmid which coils itself up; that is super coils. Supercoiling reduces the effective surface area to volume ratio culminating in less drag for a given charge (constant for the same species regardless of conformation) and thus navigates through the pores of the gel more quickly.
However, Plasmid DNA can be subject to nicks and these relax supercoiling thus opening up the DNA and causing it to move more slowly. The degree of nicking will determine how relaxed a plasmid becomes; how much increase therefore is incurred in surface area and thus how much drag is associated with electrophoresis leading to retarded migration compared to more coiled species
This retardation through opening up the vector is probably what you are seeing after staining
Moral of the story: Only cut linear vector can be sized accurately
You are right,
the dimer form migrates slower than the monomer and (depending on plasmid size and gel type) than the lin. DNA. Digesting it to completion results in the cut of both (!) resriction sites of the "single cutter" in a dimer (or one in a monomer) leading to the misinterpretation.
Determination of this can either be done by partial(!) digest with a linearizing single cutter enzyme (resulting in the expected 4.7 kb band and the intermediate product - a 9.4 kb band - in traces as well) or by CGE plasmid topology analysis.
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