Applied Techniques: DNA Isolation, Gel Electrophoresis

Objectives
At the end of the experiment each student will be able to
1) Isolate DNA from your cheek cells
2) Separate dye molecules using agarose gels and electrophoresis

Background
DNA is the “information molecule” between a positive and negative
found in all living cells. It is the electrode, the positively charged
material that gives all organisms their molecules move toward the negative
unique inherited characteristics. DNA is electrode and the negatively charged
also the “Betty Crocker Cook Book” for molecules migrate toward the positive
all the proteins an organism needs to electrode (opposites attract). Since
produce using the process of protein molecules free in solution easily
synthesis. DNA has a double-helix disperse, agarose is used to restrain the
structure, and consists of a 5-carbon diffusion of the molecules being
sugar called deoxyribose, a phosphate separated. The agarose contains large
backbone and four types of nitrogenous pores (empty spaces) through which
bases (adenine, thymine, cytosine, charged molecules can move while
guanine). DNA isolation is an important being restrained in a limited area. Thus
procedure for the molecular biologist. In DNA or dye molecules, dissolved in a
order to study the genes/DNA of any dense solution, sink into wells formed in
organism it is necessary to purify its the agarose. When electrical current is
genome and to isolate the individual applied, the molecules in the wells
genes. In the last 25 years, molecular migrate through the agarose gel towards
techniques such as sequencing DNA, the respective electrodes while
cutting out individual genes, cloning and remaining in a relatively compact band.
amplifying DNA have made molecular The smaller the molecule the easier it is
biology, biotechnology and genetic able to move through the pores and thus
engineering possible. Once isolated, the more rapidly it migrates. This results
DNA can be sequenced, manipulated in a graded (proportional) separation of
and/or altered. All living organisms molecules of different sizes from the
carry a certain volume of DNA, for well toward the electrode, with the
example; bacteria contain approximately largest molecules moving the shortest
10–14 grams of DNA per cell and the distance from the wells and the smaller
DNA from E. coli is about 1mm in molecules moving the farther distances.
length if it is stretched out.
Genetic analysis is commonplace in
Gel electrophoresis separates criminal investigations. Samples
macromolecules on the basis of size and collected from a crime scene such as
rate of movement through a gel under hair, blood, and or tissue can be
the influence of an electric field. Briefly, analyzed and a DNA fingerprint, a
when charged molecules are placed unique genetic pattern, obtained.
Background cont’d electrophoresis, the DNA sequence is
DNA samples to be tested are first “cut” transferred onto nitrocellulose paper
into many different sized fragments (blotting) and visualization of specific
using restriction enzymes. Because DNA sequences is accomplished. Since
restriction enzymes cut DNA at everyone has unique DNA patterns or
locations with specific sequences, each fingerprints, potential criminal samples
person has the potential to produce a and victim samples can be separated and
unique set of DNA fragments. After the matches can be made to determine the
fragments are separated by gel suspect and the victim.

Procedure
DNA contains the instructions for making you. How you look, what blood type you have, even
your tendency to get some diseases. It is found inside the nucleus in just about every single cell
of your body. In this activity, you'll break away the membrane around the cell and its nucleus so
that you can see your very own DNA.

1. This procedure will collect some of the buccal cells that line the inside of your mouth. These
cells are continuously being sloughed off of your cheeks. Swish 5ml 0.9 percent salt water in
your mouth for 30 seconds.

2. Spit the water into your cup. Pour this into a large test tube containing 2.5 ml of 25% liquid
detergent.

3. Cap tube and gently rock it on its side for 2-3 minutes. The detergent will break open the cell
membrane to release the DNA into the soap solution. Do not be too vigorous while mixing!
DNA is a very long molecule. Physical abuse can break it into smaller fragments, a process
known as shearing.

4. Open and slightly tilt the tube and pour about 2.5 ml of the chilled 95 percent ethanol down
the side of the tube so that it forms a layer on the top of your soapy solution.

5. Allow tube to stand for 3 minutes.

6. Use a transfer pipet to pick up the DNA along with some of the 95 percent ethanol and
transfer it to the small vial or microfuge tube. Fill the rest of the vial with the ethanol and cap
it with the cork provided.

Your DNA should remain solid in this solution.

AGAROSE GEL ELECTROPHORESIS

For this exercise, we will be separating different dyes, instead of DNA. Dyes more are visible
and less toxic than some of the reagents used in viewing DNA fragments. Because theses dyes
have different electrical charges and molecular weights, they will demonstrate different
migration patterns on the agarose gel. We will run six “known” dyes, and two “unknown” dyes,
to demonstrate the general principle involved.

Casting the gel:

1. Place the casting tray in the bottom of the gel box. Insert the dams so that the inward facing
sides are vertical. Insert the comb (8 tooth) in the center position.
2. Obtain a tube of melted agarose from the water bath. Slowly pour the agarose into the
casting try. Try to avoid getting air bubbles in the gel (If you do break them with a pipette
tip). Let the gel cool (about ten minutes). It will be firm, milky in color, and the look like
Jell-O.
3. When the gel is solidified, remove the dams. Pour electrophoresis buffer into the end
chambers so that the buffer just covers the gel. Carefully remove the comb without tearing
the gel.

Loading the gel:

1. Set the micropipette to deliver 5 micro-liters. Be sure you have read the techniques and
practiced with the water before loading the samples.
2. Push the pipette into one of the tips in the tip rack (Do not touch the tip with your fingers,
this will cause contamination of the samples) Push the plunger knob on the micropipette
down to the first stop. Keeping the plunger down, lower the pipette tip into the liquid in the
bottom of the #1 sample tube. Release the plunger to draw a sample into the pipette tip
3. Carefully place the pipette tip into the first well (hole) in the gel the well closest to the hinge
of the box lid. Try not to poke the bottom or sides of the well. It may help to steady the
pipettor with your free hand.
4. Keep your hand very steady and SLOWLY press the plunger to the first stop then the second
stop to release the sample completely If you do this too quickly the sample may shoot up out
of the well and get lost in the buffer. Before you release the plunger, remove the tip from the
buffer or else you will pick up buffer as you take the tip out of the gel.. Some slop over is OK
as long as most of the sample gets in the well.
5. Using the tip ejector button, eject the tip into the waste container on your bench.
6. Repeat this procedure for all the samples. Fill the wells in numerical order 1-8 across the gel.
Everyone in your group should have the opportunity to load a sample or two.

Running the gel:

1. When all samples are loaded, close the

lid on the gel box. Check that the power
supply is turned off before connecting it
to the gel box. Connect the red wire
from the red (+) connector (anode) on
the power source to the red connector on
the gel box. Connect the black wire
from black(-) connector (cathode) on the
power source to the black connector on
the gel box. Turn on the power supply
and set it to about 80 volts. If working
properly, you should see bubbles
forming in the buffer around the
electrodes within a couple of minutes.
2. Observe the movement of the dyes in the
gel. Electrophoresis should be complete
in 20 – 30 minutes. Stop the process before any dyes reach the end of the gel.
3. Turn off the power, unplug the wires, open the gel box. Lift the gel tray out and set it on a
paper towel on the lab table. Keep track of where #1 is in relation to the plus and minus
directions on the gel. Blot the gel onto filter paper.
4. Investigate each of 8 samples. Some will have a single band, some will have double bands.
Determine what is happening with each of the bands.
5. Pour the used buffer in one of the “Used Buffer” flasks on the demonstration table. Put the
gel in the trash (DON’T throw the gel tray away with the gel!!) Return the gel tray, dams,
and combs to the gel box.
Study Questions

1. What is the texture of the DNA you’ve isolated?

2. After running the gel why do some of the lanes have two bands?

3. Why do macromolecules migrate in the agarose gel?

4. What is the purpose of the electrical current applied during gel electrophoresis?

5. Why are dyes used instead of DNA in the class?