How to interpret DNA gel electrophoresis results (2023)

Agarose gel electrophoresis is a molecular biological method to analyze and separate DNA fragments according to their size. When using gel electrophoresis to help you with molecular cloning, you may encounter a common problem.

For example, you can excise your digested plasmid DNA from agarose. However, you see more than one gang on your open-minded sample and wonder which ones to cut out. In this article, we'll go over the different forms of plasmid DNA and offer some useful tips for interpreting your gel.

How to interpret DNA gel electrophoresis results (1)

Article content:

The structure of agarose

How does a circular plasmid DNA run during gel electrophoresis?

4 Common forms of plasmid DNA

How to interpret gel electrophoresis results

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The structure of agarose

Agarose, made from algae, is a polysaccharide agar. During polymerization, agarose polymers bond non-covalently and form a network of bundles. This network consists of pores with molecular filter properties.

(Video) Electrophoresis: How to Read Results

How to interpret DNA gel electrophoresis results (2)

Conceptual representation of agarose gel at the microscopic level.

DNA separation occurs due to the mesh-like nature of the agarose gel. Smaller fragments of DNA can move quickly through the pores while larger fragments get throughCaughtand therefore travel slowly.

Let's look at how this all works. A gel looks porous under a powerful microscope, but to the naked eye it looks like a smooth, opaque gelatin in the shape of a square withSpringnear one end of the surface.A well is a hollow pocket in the gel into which the DNA is loaded. Due to the negatively charged phosphate backbone, DNA carries a slight negative charge, which allows the molecule to migrate to the positively charged anode. The travel distance of DNA molecules within an agarose gel is proportional to the logarithm of its molecular weight.

How does a circular plasmid DNA run during gel electrophoresis?

Gel electrophoresis conditions (including the presence of ethidium bromide, gel concentrations, electric field strength, temperature, and ionic strength of the electrophoresis buffer) can affect the mobility of plasmid DNA. Due to the mesh-like nature of the agarose gel, circular plasmid DNA is more easily captured in the agarose mesh.

How to interpret DNA gel electrophoresis results (3)

Electrophoretic capture is a balance between electrophoretic force (pulling the circular plasmid DNA against the trap) and diffusion (allowing the circular plasmid DNA to escape from a trap). So large circular molecules have a greater chance of being captured than smaller DNA. Supercoiled DNA is more difficult to capture due to the small size of twisted DNA.

How to interpret DNA gel electrophoresis results (4)

4 Common forms of plasmid DNA

CCC-Monomer (Covalently Closed Circle).

CCC monomer is a negatively charged and supercoiled plasmid. Intact supercoiled plasmids have compact double-stranded DNA that is twisted on itself. Plasmid DNA isolated from bacterial hosts is usually in this CCC form. Undigested plasmid DNA is usually supercoiled.

OC (Open Circular) Monomer

An open circular shape is caused by the nicking (splitting) of a DNA strand. UV irradiation or nucleases can cause this single-strand break. This structure is a relaxed and less compact plasmid form. It also has less supercoiling than the CCC shape.

How to interpret DNA gel electrophoresis results (5)

(Video) How to understand Gel Electrophoresis results 1

Linear Monomer

The linear form is the result of restriction endonuclease cleavage of both strands of DNA.

OC-Dimer (Concatemer)

OC dimer is an oligomeric form of plasmids. Concatemer can occur due to replication. Dimers are usually twice as large compared to monomers.

How to interpret gel electrophoresis results

  1. If possible, load undigested, linearized, and UV-irradiated plasmids side-by-side in the agarose gel, then you can compare the bands between these samples.
  2. In general, monomeric supercoiled CCC forms move faster than all other forms because they have a compact supercoiled DNA structure. Therefore, they appear further down the gel.
  3. Open circular (OC) and linear monomers move slower than the supercoiled CCC monomer. They have more trouble getting through the pores in the gel matrix than the CCC form. Therefore, OC forms appear higher in the gel. The order of migration is usually the monomeric CCC form (the fastest), followed by the linear form and then the OC form.
  4. Completely digested plasmid DNA usually shows only a single band, a linear form of the plasmid, in its lane of the expected size. Undigested plasmid can have two forms appearing in its lane: CCC dimer and CCC monomer forms. The dimer forms usually move slower than the monomers due to their larger and twice the size compared to monomers. Hence it appears higher in a gel than in a monomer. The CCC monomer form runs faster than the linear form of the digested plasmid DNA.

How to interpret DNA gel electrophoresis results (6)

Gel electrophoresis examples of plasmid forms.Spur 1: DNA ladder. Lane 2: Undigested plasmid A. Lane 3: Completely digested plasmid A. Lane 4: UV-irradiated plasmid DNA.

Now, as an exercise, can you guess each plasmid shape from these bands in the agarose gel below?

Gel electrophoresis.Lane 1: DNA ladder. Lane 2: undigested plasmid A. Lane 3: fully digested plasmid A.


For track 2, you may see two tapes. The faint band at the top is OC and the bottom is the CCC shape. For lane 3, it is the fully digested plasmid, hence the band is linear in shape.

How do you identify your bands from your agarose gels?

During gel electrophoresis you may need to load into the wells uncut plasmid DNA, digested DNA fragment, PCR product and probably genomic DNA that you will use as PCR template. Your digested DNA fragment is a digested PCR product. The next step is to identify these bands to figure out which one to cut.

How to interpret DNA gel electrophoresis results (7)

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 you want to cut out.

(Video) Interpreting Gel Epectrophoresis

Tips for identifying the right band to cut out of your gel

  • Uncut plasmid DNA on the agarose gel is easy to identify as it can have two plasmid forms (OC and CCC form).
  • Digested plasmid, digested DNA fragment, PCR product, and genomic DNA may all show a single band. To identify these bands, you need to check their size using the DNA ladder. Their digested plasmid has a linear form with a size between OC and CCC forms of the uncut plasmid. Genomic DNA is large in size. So genomic DNA will usually show up at the top of your gel (very close to your well).
  • Digested DNA fragments may show a single band almost similar in size to your PCR product. This is your target size, and the band in this digested fragment of DNA is the one you want to excise.
  • At the bottom of the PCR product lane you may see a faint band indicative of small molecules. These small molecules are your primer molecules, which combine with other primer molecules to form a primer dimer. The size of these small molecules isn't your target size, so you don't want to clip that band.

      How to interpret DNA gel electrophoresis results (8)

      To learn more about interpreting DNA gel electrophoresis, watch our video below:

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      Cole, K.D. & Tellez, C.M. (2002). Separation of large circular DNA by electrophoresis in agarose gels.Biotech advances, 18th century(1), 82-87.

      Green, M. R., & Sambrook, J. (2019a). Agarose-Geleelectrophoresis.Logs from Cold Spring Harbor, 2019(1), pdb. prot100404.

      Johnson, P.H., & Grossman, L.I. (1977). Electrophoresis of DNA in agarose gels. Optimization of the separation of conformational isomers of double- and single-stranded DNAs.Biochemistry, 16(19), 4217-4225.

      (Video) Lab Review - Analyzing Gel Electrophoresis Results (Unit 12 Biotechnology)

      Schleef, M. (2008).Plasmids for therapy and vaccination: John Wiley & Sons.

      Schmidt, T., Friehs, K., & Flaschen, E. (2001). Structures of plasmid DNA.Plasmids for therapy and vaccination, 29-43


      How do you interpret the results of a gel electrophoresis test? ›

      A faint, thin band indicates that a relatively small amount of that DNA molecule is present in the sample. Lanes with 1 band may indicate that the sample contains only a single DNA molecule, while lanes with multiple bands indicate the presence of multiple molecules.

      How do you read a gel electrophoresis sequence? ›

      The bands of the gel are detected, and then the sequence is read from the bottom of the gel to the top, including bands in all four lanes. For instance, if the lowest band across all four lanes appears in the A reaction lane, then the first nucleotide in the sequence is A.

      How do you determine when the DNA has run far enough through the gel? ›

      The distance the DNA has migrated in the gel can be judged visually by monitoring the migration of the loading buffer dye. The electrical current is left on long enough to ensure that the DNA fragments move far enough across the gel to separate them, but not so long that they run off the end of the gel.

      How would you interpret results of DNA restriction analysis by gel electrophoresis if there was a difference in one band? ›

      How would you interpret results of DNA restriction analysis by gel electrophoresis if there was a difference in one band? The samples are not identical. Multiple probes bind to target sequences in: branched DNA amplification.

      What do the bands in gel electrophoresis represent? ›

      Small DNA molecules move more quickly through the gel than larger DNA molecules. The result is a series of 'bands', with each band containing DNA molecules of a particular size. The bands furthest from the start of the gel contain the smallest fragments of DNA.

      What are the 5 steps to DNA analysis using gel electrophoresis? ›

      In this manner, DNA fragments in a solution are separated on the basis of size. There are several basic steps to performing gel electrophoresis that will be described below; 1) Pouring the gel, 2) Preparing your samples, 3) Loading the gel, 4) Running the gel (exposing it to an electric field) and 5) Staining the gel.

      How do you read a DNA molecule? ›

      DNA is 'read' in a specific direction, just like letters and words in the English language are read from left to right. Each end of DNA molecule has a number. One end is referred to as 5' (five prime) and the other end is referred to as 3' (three prime).

      What is the result of gel electrophoresis? ›

      The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric field is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel.

      What disease can be detected by gel electrophoresis? ›

      Many diseases such as liver, renal pathological disorders, inflammation, proteinemia, multiple myeloma, and macroglobulinemia can be diagnosed by electrophoresis.

      What is the normal range of protein electrophoresis? ›

      Normal Results

      Normal value ranges are: Total protein: 6.4 to 8.3 grams per deciliter (g/dL) or 64 to 83 grams per liter (g/L) Albumin: 3.5 to 5.0 g/dL or 35 to 50 g/L. Alpha-1 globulin: 0.1 to 0.3 g/dL or 1 to 3 g/L.

      What does protein electrophoresis tell you? ›

      Protein electrophoresis is a test that measures specific proteins in the blood. The test separates proteins in the blood based on their electrical charge. The protein electrophoresis test is often used to find abnormal substances called M proteins.


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