They have more struggle passing through the pores in the gel matrix than the CCC form.
Open circular (OC) and linear monomers move slower than the supercoiled CCC monomer.Therefore, they will appear further down in the gel.
In general, monomer supercoiled CCC forms move faster than any other forms, because they have compact supercoiled DNA structure.If possible, load undigested, linearized, and UV radiated plasmids next to each other into the agarose gel, then you can compare the bands between those samples.Dimers are usually doubling in size when compared to monomers. OC dimer is an oligomeric form of plasmids. Linear form is a result of a cleavage on both DNA strands caused by restriction endonucleases.
It also has less supercoiling than the CCC form. This structure is a relaxed and less compact form of plasmid. UV irradiation or nucleases can cause this single-strand break. Undigested plasmid DNA are usually supercoiled.Īn open circular form is caused by the nicking (cleavage) of one DNA strand. Plasmid DNA isolated from bacterial hosts are usually present in this CCC form. Intact supercoiled plasmids have compact double-stranded DNA twisted around itself. Supercoiled DNA are more difficult to trap due to the small size of the twistedĤ Common Forms of Plasmid DNA CCC (Covalently Closed Circle) MonomerĬCC monomer is a negatively charged and supercoiled plasmid. So, largeĬircular molecules have a greater chance to get trapped than smaller DNA. The electrophoretic trapping is a balance between theĮlectrophoretic force (pulling the circular plasmid DNA against the trap) andĭiffusion (allowing the circular plasmid DNA to escape a trap). Due to the net-like nature of agarose gel, circular plasmid DNA is caught up easier in the agarose mesh. The gel electrophoresis conditions (including the presence of ethidium bromide, gel concentrations, electric field strength, temperature, and ionic strength of the electrophoresis buffer) may affect the mobility of the plasmid DNA. The travel distance of DNA molecules within an agarose gel is proportional to the log of its molecular weight. Because of the negatively charged phosphate backbone, DNA holds a slight negative charge that allows the molecule to migrate to the positively charged anode. A well is a hollow pocket in the gel where the DNA is loaded. Under a powerful microscope, a gel will look porous, but to the naked eye, it looks like a smooth, opaque gelatin in the shape of a square with wells near one end of the surface. Smaller DNA fragments can move quickly through the pores, while larger fragments get caught and therefore travel slowly. This network consists of pores with molecular filtering properties.Ĭonceptual Rendering of Agarose Gel at a Microscopic Level.ĭNA separation occurs due to the mesh-like nature of the agarose gel. During polymerization, agarose polymers link non-covalently and form a network of bundles.
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How to Interpret Gel Electrophoresis ResultsĪgarose, produced from seaweed, is a polysaccharide agar. How Does a Circular Plasmid DNA Run During Gel Electrophoresis? In this article, we review the different forms of plasmid DNA and offer some useful tips to interpret your gel. However, you see more than one band on your digested sample and you wonder which one to cut. When you use gelĮlectrophoresis to help you with molecular cloning, you may run into a common problem.įor an example, you are ready to excise your digested plasmid DNA from agarose. Agarose gel electrophoresis is a molecular biology method toĪnalyze and separate DNA fragments based on their size.