Agarose gel electrophoresis is a molecular biology method to analyze and separate DNA fragments based on their size. When you use gel electrophoresis to help you with molecular cloning, you may run into a common problem.
For an example, you are ready to excise your digested plasmid DNA from agarose. However, you see more than one band on your digested sample and you wonder which one to cut. In this article, we review the different forms of plasmid DNA and offer some useful tips to interpret your gel.
The Structure of Agarose
Agarose, produced from seaweed, is a polysaccharide agar. During polymerization, agarose polymers link non-covalently and form a network of bundles. This network consists of pores with molecular filtering properties.
Conceptual Rendering of Agarose Gel at a Microscopic Level.
DNA separation occurs due to the mesh-like nature of the agarose gel. Smaller DNA fragments can move quickly through the pores, while larger fragments get caught and therefore travel slowly.
Let’s look at how this all works. 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. A well is a hollow pocket in the gel where the DNA is loaded. Because of the negatively charged phosphate backbone, DNA holds a slight negative charge that allows the molecule to migrate to the positively charged anode. The travel distance of DNA molecules within an agarose gel is proportional to the log of its molecular weight.
How Does a Circular Plasmid DNA Run During Gel Electrophoresis?
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. Due to the net-like nature of agarose gel, circular plasmid DNA is caught up easier in the agarose mesh.
The electrophoretic trapping is a balance between the
electrophoretic force (pulling the circular plasmid DNA against the trap) and
diffusion (allowing the circular plasmid DNA to escape a trap). So, large
circular molecules have a greater chance to get trapped than smaller DNA. Supercoiled DNA are more difficult to trap due to the small size of the twisted
4 Common Forms of Plasmid DNA
CCC (Covalently Closed Circle) Monomer
CCC monomer is a negatively charged and supercoiled plasmid. Intact supercoiled plasmids have compact double-stranded DNA twisted around itself. Plasmid DNA isolated from bacterial hosts are usually present in this CCC form. Undigested plasmid DNA are usually supercoiled.
OC (Open Circular) Monomer
An open circular form is caused by the nicking (cleavage) of one DNA strand. UV irradiation or nucleases can cause this single-strand break. This structure is a relaxed and less compact form of plasmid. It also has less supercoiling than the CCC form.
Linear form is a result of a cleavage on both DNA strands caused by restriction endonucleases.
OC Dimer (Concatemer)
OC dimer is an oligomeric form of plasmids. Concatemer can occur due to replication. Dimers are usually doubling in size when compared to monomers.
How to Interpret Gel Electrophoresis Results
- 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.
- In general, monomer supercoiled CCC forms move faster than any other forms, because they have compact supercoiled DNA structure. Therefore, they will appear further down in the gel.
- Open circular (OC) and linear monomers move slower than the supercoiled CCC monomer. They have more struggle passing through the pores in the gel matrix than the CCC form. Therefore, OC forms will appear higher in the gel. The order of migration is usually the monomer CCC form (the fastest), followed by the linear form and OC form.
- Completely digested plasmid DNA usually show only a single band, a linear form of the plasmid, in its lane with the expected size. Undigested plasmid may have two forms show up in its lane: CCC dimer and CCC monomer forms. The dimer forms, due to their larger and doubling size compared to monomers, usually move slower than the monomers. Therefore, it will appear higher in a gel than a monomer. The CCC monomer form runs faster than the linear form of digested plasmid DNA.
Gel Electrophoresis Examples for Plasmid Forms. Lane 1: DNA Ladder. Lane 2: Undigested plasmid A. Lane 3: Completely digested plasmid A. Lane 4: UV-irradiated Plasmid DNA.
Now, as a practice, can you guess each plasmid form from these
bands from the agarose gel below?
Gel Electrophoresis. Lane 1: DNA Ladder. Lane 2: Undigested plasmid A. Lane 3: Completely digested plasmid A.
For Lane 2, you may be able to see two bands. The faint band on top is OC and the one below is the CCC form. For the Lane 3, it’s the completely digested plasmid, so the band has a linear form.
How Do You Identify Your Bands from Your Agarose Gels?
During gel electrophoresis, you may have to load uncut plasmid DNA, digested DNA fragment, PCR product, and probably genomic DNA that you use as a PCR template into the wells. Your digested DNA fragment is a digested PCR product. The next step is to identify those bands to figure out which one to cut.
Gel Electrophoresis. Lane 1: DNA Ladder. Lane 2: Undigested plasmid A. Lane 3: Completely digested plasmid A. Lane 4: Digested PCR product (or DNA Fragment). Lane 5: PCR Product (with a faint primer dimer band). Lane 6: Genomic DNA. The white arrows indicate the bands that you want to excise.
Tips to Identify the Correct Band to Excise from Your Gel
- Uncut plasmid DNA on the agarose gel is easy to identify because it may have two forms of plasmid (OC and CCC forms).
- Digested plasmid, digested DNA fragment, PCR product, and genomic DNA may all have one single band. To identify these bands, you will have to check on their size by consulting the DNA ladder. Your digested plasmid has a linear form with the size in between OC and CCC forms of the uncut plasmid. Genomic DNA has a large size. So, the genomic DNA usually show at the very top of your gel (very close to your well).
- Digested DNA fragment may have a single band at almost similar size with your PCR product. This is your target size, and the band in this digested DNA fragment is the one you want to excise.
- At the bottom of the PCR product lane, you may see a faint band indicating small molecules. These small molecules are your primer molecules that link to other primer molecules to form a primer dimer. The size of these small molecules is not your target size, so you don’t want to excise this band.
Agarose LE (Molecular Biology Grade) (Catalog No. A-201)
High Resolution Agarose (For Nucleotides < 1kb) (Catalog No. A-202)
Low Melt Agarose (Catalog No. A-204)
1 kb DNA Ladder (Catalog No. D010)
1 kb PLUS™ DNA Ladder (Catalog No. D011)
100 bp DNA Ladder (Catalog No. D001)
100 bp PLUS™ DNA Ladder (Catalog No. D003)
50 bp DNA Ladder (Catalog No. D100)
VersaLadder™, 100-10,000 bp (Catalog No. D012)
Gel Loading Dye Products
6X Blue Loading Dye (Catalog No. L002)
6X GelRed™ Prestain Loading Buffer with Blue Tracking Dyes (Catalog No. G-730)
6X GelRed™ Prestain Loading Buffer with Orange Tracking Dye (Catalog No. G-735)
6X Green Loading Dye (Catalog No. L001)
Bromophenol Blue Free Acid, ACS Grade (Catalog No. B092)
Xylene Cyanol FF, Ultra Pure (Catalog No. X-300)
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Johnson, P. H., & Grossman, L. I. (1977). Electrophoresis of DNA in agarose gels. Optimizing separations of conformational isomers of double-and single-stranded DNAs. Biochemistry, 16(19), 4217-4225.
Schleef, M. (2008). Plasmids for therapy and vaccination: John Wiley & Sons.
Schmidt, T., Friehs, K., & Flaschel, E. (2001). Structures of plasmid DNA. Plasmids for therapy and vaccination, 29-43