Teacher's Section: DNA Analysis by Agarose Gel

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DNA Analysis by Agarose Gel

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Using Genetic Evidence
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DNA Analysis by Agarose Gel Electrophoresis

Gel electrophoresis is an important molecular biology tool. Gel electrophoresis enables us to study DNA. It can be used to determine the sequence of nitrogen bases, the size of an insertion or deletion, or the presence of a point mutation; it can also be used to distinguish between variable sized alleles at a single locus and to assess the quality and quantity of DNA present in a sample.

Gel electrophoresis is a method of separating chemical compounds and molecules by their size and charge. The substances being separated are placed in wells in an agarose gel and subjected to an electrical field. Negatively charged molecules move towards the positive anode, and positively charged molecules move towards the negative anode. Longer or larger molecules have difficulty traveling through the gel; they become entangled in the gel matrix. Shorter or smaller molecules migrate through the agarose matrix faster and thus travel farther in a given time period. Similar sized fragments travel at relatively the same speed and form a tight "band" when stained.

In these activities, we will introduce the concept of gel electrophoresis and use it to determine the quantity and quality of the DNA the students extracted. Electrophoresis also can be used in DNA sequencing, profiling or "fingerprinting", and genetic engineering.

Overview of Activities

The activities are divided into three parts followed by a student lab sheet:

1. Preparing an Agarose Gel

2. Loading DNA Samples and Running an Agarose Gel

3. Staining the DNA in an Agarose Gel

Quantification of DNA Using An Agarose Gel - Student Lab Sheet

Each portion can be accomplished in a 50-55 minute time period. If time is short, you can prepare the gels (Activity 1) and stain the gels (Activity 3) for the students.

Preparing an Agarose Gel

Different types and concentrations of media can be used to make a gel. The concentration and type of media will affect the gel's pore size and ability to separate similarly sized fragments. Agarose gels separate DNA fragments differing by a hundred or more base pairs, while polyacrylamide gels can separate DNA fragments differing by a single base pair. Well forming combs are inserted into the gel media as it cools and solidifies. After the gel solidifies, the combs are removed. The gel will be placed into an electrophoresis chamber, covered with a buffer solution, and DNA samples will be loaded directly into the wells.

Preparing an agarose gel involves melting a specified amount of agarose in TBE buffer, cooling the solution, and pouring it into the gel casting tray. Gels solidify in 15-20 minutes. They can be prepared by you ahead of time or students may prepare their own (see Preparing an Agarose Gel). Gels made ahead of time (1-2 days maximum) should be wrapped in plastic wrap and stored in the refrigerator. Students do a great job preparing gels, but sometimes it is easier and quicker to prepare them yourself, especially if you have a limited number of balances, microwaves, etc., or large classes. Advanced students or student aides can also be assigned this task.

Loading an Agarose Gel

Gels are covered with a buffer solution. Prior to loading the samples, the DNA must be mixed with a loading dye. The loading dye serves two purposes: first, it increases the density of the DNA so it will sink into the wells, and second, it provides a visual marker so you know how far the DNA (which is not visible) has traveled in the gel. Concentration and size standards are loaded for comparison. Each DNA sample should be loaded into a different lane (see Loading and Running an Agarose Gel).

Students have a tendency to stick the micropipettor into the well while loading their samples. This causes two problems: first, they can poke a hole in the bottom of the gel (their sample will drain out the bottom into the buffer tank), and second, they create so much turbulence within the well that the DNA sample squirts out the top of the well. Remind them to keep the pipettor above the well, but not in the well.

You may want to let students practice using the pipettors and loading small volumes into the wells. Make practice gels using agar (23g of agar per liter of distilled water) not agar. Agar is much less expensive but cannot be used for running gels. The agar should be melted, cooled to 55 C and then poured into petri dishes. Suspend the combs in the petri dishes. Flood the practice dishes with water. Students can practice loading colored dyes mixed with glycerol or karo syrup. Common laboratory dyes, such as crystal violet, methylene blue, safarin orange, methyl orange, etc. make great practice dyes. Mix small amounts (1-2 mL) with an equal volume of karo syrup or glycerin.

Running an Agarose Gel

The buffer solution contains ions which conduct electrical charges. The charges travel through the gel because it also contains ions (it was prepared with the same buffer). DNA is negatively charged and migrates toward the positive anode. The speed with which the DNA moves through the gel is determined by the size of the DNA fragment and the voltage. DNA will migrate faster at higher voltages although lower voltages provide better resolution between similar sized fragments. (The bands are less blurry.)

Students should run their gels for about 45 minutes or until the dye travels 1/2 - to - 3/4 of the way down the gel. They will have enough time to load and run their gels in one class period if they have had time to practice setting up the gel electrophoresis equipment, loading samples into agar petri dishes, know how to prepare their DNA and the agarose gels are ready to use. Otherwise, they can continue to run their gels after the class is over if you remember to check the gels and turn them off after about an hour (total running time). Students can tell a gel is running by looking for bubbles coming from the electrodes (once the power is on). They should never touch the gel boxes while the power is on because they can electrocute themselves if the top of the tank is open. Most buffer tanks have locking tops that prevent this.

Students should also load standards in order to determine the quantity of DNA they extracted. These are DNA samples of known concentrations that can be used for comparison. Typical standards include: 50 ug/uL, 100 ug/uL and a 1 Kb ladder. The band produced by the 100 ug/uL sample will be twice as dark as the band produced by the 50 ug/uL sample. A 1 KB ladder produces numerous bands of several different sizes. Concentration standards and ladders can be ordered from molecular biology companies.

Staining the DNA in an Agarose Gel

DNA must be stained to be visible. Scientists use ethidium bromide because it is highly sensitive to DNA and is visible under ultraviolet light. Ethidium bromide is a mutagen and a carcinogen so it should only be used with extreme care. Methylene blue, a dye, may also be used to stain DNA. It is not harmful but requires the presence of higher quantities of DNA to be visible (see Staining the DNA in an Agarose Gel). Methylene blue will stain clothes, hands, and equipment. Spills should be wiped up immediately and flushed with water.

The gels stain the best if they are in plastic container slightly bigger than the gel itself. This also conserves the amount of stain you use. Flood the gel with the stain. Gently rock the container every 10 minutes to move the dye across the gel. Students can do this section at the same time they are doing something else.

Study the gel and interpret the results:

A single band near the wells is characteristic of whole, uncut genomic DNA.
A smear of DNA down the lane represents large pieces of genomic DNA that are partially degraded (broken down) into thousands of smaller fragments or "cut" genomic DNA.
Discrete bands represent a group of DNA fragments which are similar in length.
Darker bands have more DNA
No bands are present when the concentration is either very low or DNA is not present. This may occur if students punctured the well while loading their DNA.

The University of Arizona
Department of Biochemistry and Molecular Biophysics
General Biology Program for Secondary Teachers
warder@email.arizona.edu