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Agarose Gel Electrophoresis

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What is Agarose Gel Electrophoresis?

Polymerase Chain Reaction (PCR)

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Agarose Gel Electrophoresis: What is it?

Electrophoresis is a method developed in the 1930s that led to Arne Tiselius’ Nobel Prize in Chemistry.
It is used to separate mixtures of DNA, RNA, or proteins according to their molecular size and charge.
The “agarose” in Agarose gel electrophoresis specifies the gel type used is agarose (as opposed to other types like Polyacrylamide).
Agarose Gel electrophoresis is a tool/method for determining:

DNA fragment size
Concentration of DNA
Purifying DNA

Agarose Gel Electrophoresis

What is it?

Learn more about gel electrophoresis by watching the video below:

Introduction to Agarose Gel Electrophoresis Video

In the 1930s, Arne Tiselius developed an apparatus that used electrical charges to separate molecules in a complex mixture over a sheet of damp paper. This process is called electrophoresis, which involves using an electrical charge to separate substances from one another. This is only possible because, even though molecules may have the same charge, they vary in size, shape, and mass. Tiselius received a Nobel Prize in Chemistry for this invention.

In the 1970s, researchers optimized Tiselius' invention and adapted the electrophoresis to a gel matrix instead of a damp paper. This change revolutionized science and allowed the separation of molecules, such as DNA, RNA, and proteins, using their intrinsic properties: charge and size.

Agarose is one of these gel matrices and specifies the chemical component responsible for forming the gel. So, now, when you listen to Agarose gel electrophoresis, you know that this is a process that uses an electrical charge to separate molecules in an agarose gel matrix. A different type of gel matrix is polyacrylamide. The agarose gel separates better nucleic acids (DNA and RNA), whereas the polyacrylamide works best for proteins. By using agarose gel electrophoresis, scientists can estimate the size or concentration of DNAs accurately and even obtain highly pure DNA molecules.

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How does Agarose Gel Electrophoresis work?

Agarose Gel Electrophoresis

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How Agarose Gel Electrophoresis works:

Because Nucleic acid polymers like deoxyribonucleic acid (DNA) have a negatively charged phosphate backbone, scientists realized they could cause these negatively charged molecules to move towards a positively charged electrode when a current was applied

The negatively charged DNA backbone is pulled through the gel by electrostatic forces towards the cathodic electrode.

Agarose Gel Electrophoresis: How does it work?

Before we answer the question itself, let’s recapitulate some very important concepts about DNA. DNA is a polymer made of a sugar molecule (deoxyribose), four different bases (A, C, T, and G), and a phosphate backbone. The phosphate backbone is exposed outwards when the DNA forms the double-helix by pairing the bases with a complementary DNA strand. The important characteristic of the phosphate group is that they are negatively charged molecules. And as you know, negative charges are attracted to positive charges. In summary, scientists developed agarose gel electrophoresis to separate DNA by taking advantage of the negatively charged phosphate backbone. The agarose gel is placed on a tank with a negative pole, anode, and a positive pole, cathode. An electric current will move the DNA molecules from the anode to the cathode using electrostatic forces.

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How Agarose Gel Electrophoresis works:

DNA samples are placed in a semi-solid gel that looks like Jell-O which is then placed between two electrodes
The gel is made from agarose, a substance extracted from seaweed
The gel forms a molecular mesh that biomolecules like nucleic acids can travel through
The molecular mesh is made up of long strings of polysaccharides held together with hydrogen bonds that form pores and channels

Agarose Gel Electrophoresis: How does it work?

Agarose gel

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How Agarose Gel Electrophoresis works:

As the nucleic acids are pulled toward the positively charged electrode, they bump into and get caught up by the agarose mesh.
The larger the nucleic acid, the more it bumps into and gets hung up by the agarose molecules. This results in the large molecule moving more slowly toward the positively charged cathode
The smaller nucleic acid molecules don’t get caught up as much in the agarose mesh and so they move more freely and migrate faster toward the cathode.

Agarose Gel Electrophoresis: How does it work?

a) A mix of DNA molecules of different sizes is initially at the same spot in the gel.

b) Once the electric current is applied, smaller DNA molecules rapidly move through the agarose mesh while the larger DNA molecules bump into the agarose polymers, slowing them.

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Setting up an Agarose Gel Experiment:

There are four things you need to set up an agarose gel electrophoresis experiment:

A gel tank with alternate electrodes (cathode & anode) on each end so that an electric current can be applied to “pull” the negatively charged DNA molecules towards the cathode (+).
a buffer containing ions (charged atoms) is made and poured into the tray. This allows the current to move from the anode (-) to the cathode (+). Without any ions, the electrons in the current won’t be able to move between electrodes. Typically, the buffer is a 1x TAE buffer.

Agarose Gel Electrophoresis: How does it work?

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Setting up an Agarose Gel Experiment:

Agarose gel which will be place in the middle of the tray (gel tank), and covered by the TAE buffer. To make the gel, pour a hot agarose mixture into a gel tray with rubber stoppers on each end to contain the liquid until it solidifies. You must also place a comb in the gel tray to form the wells you will pipette the DNA into.
a sample that contains the DNA molecules you want to analyze or view, and a DNA ladder. The DNA sample can be extracted from a cell, or created during a DNA creation experiment.

Agarose Gel Electrophoresis: How does it work?

Anode (-)

Cathode (+)

Agarose gel

Comb

It Is important to mention that the gel is polymerized (or synthesized) outside the electrophoretic tank. We commonly use a square (or rectangular) tray in which a hot agarose-containing solution is poured and allowed to cool down until it forms a semi-solid gel (very similar to how we prepare Jell-O at home). When the solution is still hot, we place a comb at the tray to make sure the gel will polymerize with wells at one end. Upon polymerization, the comb will be carefully removed to avoid causing any damage to the formed wells. These wells will be placed at the anode (negative pole), and this is where the DNA samples will be inserted. After everything is set, you will need your DNA sample as well as a DNA ladder to be used as a reference of size and concentration. This DNA sample could be extracted from humans, microorganisms, or even created in a laboratory.

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Running an Agarose Gel Experiment:

To run DNA samples in a gel, you pipette them into small rectangular indentations on the anode (-) side of the gel called “wells."
When all the samples are in the wells, a power supply connected to the tank in which the gel is is turned on and an electric field begins pulling the negatively charged DNA molecules through the gel matrix towards the cathode (+).
Scientists will often add a mix of DNA molecules of known molecular weights called a DNA ladder or 1 Kb ladder to one of the wells as a reference point.

Agarose Gel Electrophoresis: How does it work?

Power supply

Wells

Agarose gel

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Running an Agarose Gel Experiment:

Since smaller DNA molecules can race through the gel mesh towards the cathode leaving larger ones behind, this creates a distinction in their location on the gel based on sizes of the DNA.
The distinction happens after the current is applied for a certain time, “running a gel”
This distinction is seen in the form of “bands” on the gel. On the image on the right, each white rectangle is a band of DNA.
The bands can be bright or faint depending on the amount of DNA present at the beginning of the gel.
Sometimes the bands are blurry. This can be because the buffer used in the experiment is a bit old or because there are DNA fragments of similar size that are overlapping each other.

Agarose Gel Electrophoresis: How does it work?

Anode (-)

Cathode (+)

Bands of DNA

DNA Ladder

Wells

As mentioned before, smaller DNA fragments can easily find the pores in the gel matrix, which makes them race through the gel faster than bigger fragments. This “DNA race” will separate molecules based on their size, letting the smaller ones migrate longer than the big ones, creating a ladder-like image. When the electrical current is applied to the system, the DNAs in the wells start to “run,” like in a race where you know who will win. Each fragment will form a rectangular bright “band” on the gel. The brightness of the band indicates DNA concentration, in which brighter bands contain more DNA than fainter ones. Sometimes, a not-so-well-defined band could catch your eye on a gel. This kind of band can form a blurred image due to some reasons: your buffer environment may not be working great (the solution could be very old), or your DNA sample is enriched in fragments close in size that cannot be separated by this method, and you see an overlap.

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Running an Agarose Gel Experiment: Pro tip

When dispensing the sample into the wells, make sure that the pipette tip hovers at the top of the well. If your pipette tip is stabbing the gel at the bottom of the well, you've gone too deep.

To make sure your pipette tip is in the correct position, try moving the entire gel by pushing on one side of the well. If the gel moves, your pipette is correctly hovering at the top of the well. You can also ask a lab mate to guide you as they watch from the side.

Agarose Gel Electrophoresis: How does it work?

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Viewing your Agarose Gel results

Before loading DNA samples in the gel, they are mixed with a visualizing dye. (If you are using an Amino Labs kit, this step is often already done for you!)
The dye allows the DNA in the gel to fluoresce under blacklight or in a gel imaging system.
Once the gel has run for sufficient amount of time and the DNA samples have been separated, the gel is removed from the tank and viewed under a UV light to analyze your results.
Taking a photo of the gel and enhancing the brightness, contrast or exposure time can help with viewing the bands clearly.

Agarose Gel Electrophoresis: How does it work?

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DNA Ladders in gel electrophoresis

Agarose Gel Electrophoresis

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DNA Ladders in gel electrophoresis

The DNA ladder is a valuable tool scientist use in their gel electrophoresis experiment to estimate the size of the DNA fragments or mixture they are running through the gel.
The DNA ladder contains DNA fragments of known lengths. It is similar to a ruler you would use to measure paper or wood when you build something new, or like a scale you can use to weight ingredients when you cook. The ruler, scale and DNA ladder are all reference tools you can use to achieve a goal.

DNA Ladder in a gel

Agarose Gel Electrophoresis: DNA Ladders

Reference DNA Ladder

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DNA Ladders in gel electrophoresis

Agarose Gel Electrophoresis: DNA Ladders

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DNA Ladders in gel electrophoresis

Agarose Gel Electrophoresis: DNA Ladders

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DNA Ladders in gel electrophoresis

Agarose Gel Electrophoresis: DNA Ladders

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DNA Ladders in gel electrophoresis

Agarose Gel Electrophoresis: DNA Ladders

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Troubleshooting Group Activity

Agarose Gel Electrophoresis

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Agarose Gel Electrophoresis: Troubleshooting activity

After loading your gel with your samples, you start your run but notice that the samples are not migrating towards the cathode. What could have happened?

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Agarose Gel Electrophoresis: Troubleshooting activity

The issue could be with the buffer the gel is placed in. If water was used instead of the TAE, there will not be enough conductivity for the electric current and so the DNA will not migrate

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Agarose Gel Electrophoresis: Troubleshooting activity

After running a gel you go to visualize but you notice that the DNA bands appear quite smeared. What could have gone wrong?

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Agarose Gel Electrophoresis: Troubleshooting activity

This could be a gel polymerization issue. The gel may have not been given enough time to properly solidify. It is also possible that the gel was run at a voltage that might be too high; lowering the voltage would also help provide clearer bands

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Agarose Gel Electrophoresis: Troubleshooting activity

You run an agarose gel but when you place the gel in UV light you do not see any DNA bands. What could have gone wrong?

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Agarose Gel Electrophoresis: Troubleshooting activity

It it likely that the RedSafe was not added to the warm agarose mixture. Without it, the DNA fragments cannot “light up” under UV light and will not be visible

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Glossary

Agarose Gel Electrophoresis

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Gel Electrophoresis glossary

1 kB Ladder is a mixture of DNA molecules of different sizes that can be used as a reference when determining the size of DNA samples.
1X TAE is a concentration of 1X dilution of tris-base, acetic acid and the surfactant EDTA.
Agarose is a polysaccharide that is extracted from red seaweed.
Casting a gel refers to mixing and boiling agarose powder and TAE buffer then pouring it into a tray to solidify. A comb is placed in the hot agarose liquid to form wells.
Gel Electrophoresis is a size/charge based technique that uses an electric current to separate molecules.

Agarose Gel Electrophoresis: Glossary

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Take the intro to Agarose Gel Quiz

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Conclusion

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