Agarose Gel Electrophoresis for DNA Analysis

Gel electrophoresis is a technique used to separate biological molecules based on size by applying a current to them.  The resulting size and fragment distribution pattern can often reveal useful information about the sequence of DNA bases. These patterns are sometimes called a DNA fingerprint and can be used in DNA profiling to identify the individual or species to which the DNA belongs.

Here we will cover the what gel electrophoresis content, for procedural information take a look at  ‘How to run a DNA Gel Electrophoresis experiment.

 


TL; DR

  • DNA gel electrophoresis takes fragments of DNA in a gel and uses a current to separate the fragments according to size.
  • Before a gel electrophoresis experiment, samples been to be collected by DNA extraction and prepared using PCR and restriction enzyme digests.
  • Results from electrophoresis can be used as to compare known and unknown DNA samples.


Before Gel Electrophoresis

Preparing your DNA Sample

Gel electrophoriesis is a pretty standard procedure used in almost every biology laboratory. It has broad applications for answering different questions in biology, but it typically comes to mind when we want to comparing two or more DNA samples.   

Once you’ve determined that gel electrophoresis is the right technique to answer your question, you still need to do a couple of things before you can get going. Samples first need to be collected and processed before the DNA of interest can be used for gel electrophoresis. The following steps indicate the order in which DNA is prepared:

    1. Collect samples that contain DNA – (blood, sweat, saliva, tissues)
    2. DNA Extraction – to isolate the DNA from the rest of the cell. 
    3. Polymerase Chain Reaction (PCR) – to ensure we have enough DNA to work with.
    4. Restriction Enzyme Digest -incubating our amplified DNA with restrictions enzymes will create DNA fragments. Take a moment to read about restriction enzymes if you’re unfamiliar with them.
    5. Our sample of DNA fragments is now ready for gel electrophoresis!

Link to Learning

This process of comparing DNA samples to determine if two samples contain the same DNA is called DNA profiling. It can be used in situation like determining whose DNA was at a crime scene or paternity tests.


 

Electrophoresis Setup

Now that our DNA samples are ready for gel electrophoresis, we need to set up our electrophoresis chamber. To do this we’ll need the following materials

    • 1x TAE Buffer
    • This buffer is important for things to work well. It is used both as the liquid component to make our gel and to submerege in the chamber.  It can be purchased as a concentrated stock (usually 50x) but can also be made from scratch.
    •  
    • Agarose
    • This sugar is used as the solid component of the gel. It’s mixed together to make a gel that generally around 1% agarose $\frac{mass (g)}{volume (mL)}$. This means, to make a 1% agarose gel, you would need to dissolve 1g of agarose into 100mL of 1x TAE buffer.
    •  
    • Gel Casting Tray
    • The casting tray creates the gel mold. It will come with a comb, which is used to create inserts in the mold where we will load our DNA samples.
    •  
    • Pipette & Tips
    • Pipettes and pipette tips are necessary to handle the small volumes in a precise and sterile way.
    •  
    • Electrophoresis Chamber
    • The agarose gel will sit in the electrophoresis chamber and the chamber will be filled with 1x TAE buffer. At each end of the chamber are electrodes. When they current is applied, it will travel from the anode to the cathode through the salty 1x TAE buffer. As it does so, the DNA will appeared to be ‘pushed’ towards the positive electrode.
    •  
    • Power Supply
    • The electrodes from the chamber attach to the power supply which supplies the current.
    •  

 

What’s covered here is just an overview, to see what this looks like step-by-step, take a look at how to prepare a gel electrophoresis experiment.

Steps for setting up a gel electrophoresis experiment
 Author/Source: OpenStax | License: CC BY 4.0

You may have guessed from the suffix -ose that it’s a sugar. It’s a fairly large polysaccharide derived from seaweed.  The agarose serves two purposes in this application:

  1. It gives the gel a solid form that we can place the DNA into.
  2. In the gel itself, the molecules of the sugar create a matrix that the DNA has to navigate around. Larger molecules face more resistance navigating around these molecules and therefore travel slower through the gel.

 

Running the Gel


At this point you have your experiment setup looks this this:

      • ✔ Agarose gel in the electrophoresis chamber.
      • ✔ 1x TAE buffer in the chamber submerging the agarose gel.
      • ✔ DNA samples loaded into each of the wells.
      • ✔ Electrophoresis chamber connected to your power supply.
      • ✔ Power supply plugged into a work outlet!


Samples are loaded into sample wells on the side of the gel closest to the negative electrode. Once the power supply is turned on, the negatively charged DNA will being to move through the gel, towards the positive electrode. The agarose matrix impedes the movement of larger molecules, whereas smaller molecules pass through more easily. Therefore, the distance of migration is inversely correlated to the size of the DNA fragment, with smaller fragments traveling faster. Sizes of DNA fragments can be estimated by comparing them to known sizes in the DNA ladder run on the same gel.

Steps for doing a gel electrophoresis experiment
 Author/Source: OpenStax | License: CC BY 4.0

Viewing your results


In order to you can visualize your result your gel needs to be stained. The stain is solution that interacts with DNA in such a way that allows you to see it. Some stains are applied after the gel is run, but more commonly stains are actually added to the gel during the molding process. One very common method is ethidium bromide, which inserts itself into the nucleic acids at non-specific locations. In order to see your DNA, you need to expose the gel to ultraviolet light. However, ethidium bromide is a known carcinogen and alternative stains that are much safer are now available.

DNA gel electrophoresis chamber setup.
 Author: Genome Research Limited | Sourceyour genome
| License: CC BY 4.0

The ladder is a sample of DNA fragments of know lengths. It’s provides an way to determine the general size of the fragments in you experimental samples and is usually loaded in the first and/or last lanes.

DNA Gel Electrophoresis FAQs & Review

DNA gels are made from a polysaccharide found in seaweed called agarose. Agarose gels satisfy a range of characteristics including physical and thermal stability. Another important feature is the pores that are created when the sugars solidify into gel. This matrix provides a structure that allows for DNA molecules of varying sizes to be separated, since larger molecules will take longer to move through the matrix.

The wells hold the DNA sample in place. During electrophoresis, the DNA will move into the gel, towards the positive electrode.

DNA’s negative charge comes from the presence of phosphates in its sugar-phosphate backbone. The single boned oxygen atom in PO4 is very likely to donate its hydrogen ion, hence DNA acidic property. This leaves that oxygen with a lone pair of electrons and results in DNA carrying a net negative charge.

We know that DNA has a net negative charge. In our electrophoresis chamber we have a positive electrode (cathode) and a negative electrode (anode).  Therefore, the negatively charged DNA will move towards the cathode.

Key Terms

    • Agarose
    • DNA Ladder
    • Electrophoresis
    • TAE Buffer

Complete the Gel

You were expecting to see four bands in Lane 1 of your gel. Take the bands (to the right of the gel) and place them in the places you expect to see them. You believe there the bands should be at the following locations:

      • → 652 bp
      • → 589 bp
      • → 48 bp
      • →12 bp

         

 

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  2. Clark MA, Douglas M, Choi J. “17.1 Biotechnology.” Biology 2e. OpenStax, 2018. Houston, TX. https://openstax.org/books/biology-2e/pages/17-1-biotechnologyLicenseCC BY 4.0 | License Terms: Edited & Adapted | Access for free https://openstax.org/books/biology-2e/pages/1-introduction.