Biology Lab
Name:___________________________________ Date:___________
In the 1960s, scientists discovered that bacteria have enzymes that cut, or "digest," the DNA of
foreign organisms and thereby protect the cells from invaders such as viruses. Scientists have
now isolated several hundred of these enzymes, known as restriction enzymes, or restriction
endonucleases. Each is able to recognize and cut at a specific DNA sequence, known as a
recognition sequence.
The discovery of restriction enzymes made genetic engineering
possible because researchers could use them to cut DNA into
fragments that could be analyzed and used in a variety of procedures.
In this part of the laboratory, you will use gel electrophoresis to
separate samples of DNA that have been digested by restriction
enzymes. Then you will compare fragments of unknown size to
fragments of a known size to calculate the unknown fragment sizes.
Let's begin by looking at how restriction enzymes work.
Like all enzymes, restriction enzymes are highly specific. They cut DNA only within very precise
recognition sequences. Study the illustrations below to see three different recognition
sequences. The red line shows where the enzymes will cut the DNA. Notice that all of these
recognition sites are symmetrical, or what is called "palindromic." This means that the
recognition sequence on one DNA strand reads in the opposite direction on the complementary
strand.
EcoRI cuts the DNA so that
the fragments have singlestranded ends. These are
called "sticky ends" because
they will hydrogen bond with
other strands of DNA that
have a complementary
sequence of bases. This
makes them useful in
producing recombinant DNA.
PstI cuts a different
recognition sequence, but
also produces DNA
fragments with sticky
ends.
SmaI cuts the DNA in
such a way that no
single-stranded piece is
formed. This results in
what are called "blunt
ends."
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
p 1/15
Next let's look at the laboratory procedures for cutting and separating DNA fragments.
Cutting DNA with Restriction Enzymes
You begin by mixing DNA with one or more restriction
enzymes in a small plastic microcentrifuge tube. The total
volume of the mix is about 20 µl.
Microscale Quantities
We use very small quantities
when working with DNA, so
the volumes and tools are
adapted to this microscale. In
the metric system, the prefix
"micro-" indicates onemillionth. It is symbolized by
µ, the Greek letter mu. Some
examples are:
Most restriction enzyme reactions are incubated at 37°C for
one hour. After incubation, you can analyze the DNA or use it
in other kinds of reactions, such as the bacterial
transformation you did in the first part of this lab.
1 ml = 1000 µl (1000
microlitres)
1 mg = 1000 µg (1000
micrograms)
In the next procedure, you will see how to analyze separate
DNA fragments with gel electrophoresis.
Gel Electrophoresis
Gel electrophoresis is a procedure that separates molecules on the basis of their rate of
movement through a gel under the influence of an electrical field. The direction of movement is
affected by the charge of the molecules, and the rate of movement is affected by their size and
shape, the density of the gel, and the strength of the electrical field.
DNA is a negatively charged molecule, so it will move toward the positive pole of the gel when a
current is applied. When DNA has been cut by restriction enzymes, the different-sized
fragments will migrate at different rates. Because the smallest fragments move the most
quickly, they will migrate the farthest during the time the current is on. Keep in mind that the
length of each fragment is measured in number of DNA base pairs.
In your laboratory, you will prepare and "run" your own gel electrophoresis.
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 2/15
Design of the Experiment II
In your laboratory you will be given three samples of DNA obtained from a virus, the
bacteriophage lambda. One sample will be uncut DNA, one will be incubated with the restriction
enzyme HindIII, and one will be incubated with EcoRI. You will separate the fragments of DNA
by electrophoresis, stain the DNA for visualization, and determine the fragment sizes formed in
the EcoRI digest.
The figure below is an overview of the procedure, using generalized DNA samples. Over the
following several pages we look at the procedure step by step.
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 3/15
Preparing the Gel
After making the gel, place it in an electrophoresis chamber and add buffer to cover the gel.
Be sure the wells are placed at the negative electrode end.
The apparatus is now ready to load. Do not turn on the electricity yet!
Loading the Gel
Since we cannot see DNA without special dyes, we add a tracking dye to each reaction tube.
This does not stain the DNA, but because of their size, the dye molecules move at a rate that is
similar to that of the DNA. Sucrose or glycerol is also added to the mixture to make it dense
enough to sink into the well.
Use a micropipette to load 5–10 µl from each reaction tube into a well. (Your instructor may
vary this procedure and have you load the wells with DNA before you pour the buffer.)
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 4/15
Electrophoresis
Place the top on the electrophoresis chamber and connect the electrical leads to the gel. Double
check that the wells are at the negative electrode.
When you turn on the power, the DNA/tracking dye combination will begin to move toward the
positive electrode.
Staining and Photographing the DNA
After electrophoresis, you must stain the DNA for visualization. You submerge the entire gel in
methylene blue, which will bind to the DNA. You then rinse the gel repeatedly with water, so
that the dye washes off the gel. The DNA will appear as blue bands that are easily seen when a
light is passed through the gel.
After staining the gel, photograph it for analysis.
Analysis of Results II
Each fragment of DNA is a particular number of nucleotides, or base pairs, long. When
researchers want to determine the size of DNA fragments produced with particular restriction
enzymes, they run the unknown DNA alongside DNA with known fragment sizes. The known
DNA acts as a marker. In your laboratory, the DNA that has been cut with HindIII is the
marker; you will use it to help you determine the fragment sizes in the EcoRI digest. On the
next pages we go through the procedure using HindIII and two generalized DNA samples.
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 5/15
Making a Standard Curve for HindIII DNA Fragments
If you know the fragment sizes in the HindIII digest, how do you determine the fragment sizes
in an unknown sample? You use data from the marker to prepare a standard curve, which will
provide a standard for comparison to the unknown fragment sizes. Using a standard to estimate
an unknown is sometimes called "interpolation"; you will interpolate the size of the unknown
fragments.
You begin by making a standard curve for the known sample, DNA plus HindIII. Measure the
distance each HindIII fragment migrated on the gel and then complete the chart. It is very
difficult to get exact numbers as you read this graph. If your response is in a close range, that
is acceptable.
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 6/15
Making a Standard Curve
When the data obtained for the marker
DNA is plotted on semilog graph paper,
it is an almost straight line. This is the
standard curve.
Hint: Be very careful when working
with log-based numbers. Small
mistakes in reading from the y-axis
translate into big mistakes in
determining base-pair lengths.
In your lab, you will use the standard curve to determine the fragment sizes of the EcoRI
digest. On the next two pages, you practice the procedure using two samples with unknown
fragment sizes, DNA I and DNA II.
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 7/15
Practice Problem 1
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 8/15
Practice Problem 2
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 9/15
Lab Quiz II
1. Which of the following statements is correct?
a .Longer DNA fragments migrate farther than shorter fragments.
b Migration distance is inversely proportional to the fragment size.
c. Positively charged DNA migrates more rapidly than negatively charged DNA.
d. Uncut DNA migrates farther than DNA cut with restriction enzymes.
2. How many base pairs (bp) is the fragment circled below?
a. 1200
b. 3000
c. 1500
d. 2500
e. 12,000
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 10/15
3. An instructor had her students perform this laboratory beginning with setting up their own
restriction enzyme digests. One team of students had results that looked like those below.
What is the most likely explanation for these results?
a. The students did not allow enough time for the electrophoresis separation.
b. The agarose prepartion was faulty.
c. The methylene blue did not stain the DNA evenly.
d. The restriction enzyme EcoRI did not function properly.
e. The voltage was set too low on the apparatus.
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 11/15
4. Below is a plasmid with restriction sites for BamHI and EcoRI. Several restriction digests were
done using these two enzymes either alone or in combination. Use the figure to answer questions
4–6.
Hint: Begin by determining the number and size of the fragments produced with each enzyme.
"kb" stands for kilobases, or thousands of base pairs. Which lane shows a digest with BamHI
only?
a. I
b. II
c. III
d. IV
e. V
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 12/15
5. Below is a plasmid with restriction sites for BamHI and EcoRI. Several restriction digests were
done using these two enzymes either alone or in combination. Use the figure to answer questions
4–6.
Hint: Begin by determining the number and size of the fragments produced with each enzyme.
"kb" stands for kilobases, or thousands of base pairs. Which lane shows a digest with EcoRI only?
a. I
b. II
c. III
d. IV
e. V
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 13/15
6. Below is a plasmid with restriction sites for BamHI and EcoRI. Several restriction digests were
done using these two enzymes either alone or in combination. Use the figure to answer questions
4–6.
Hint: Begin by determining the number and size of the fragments produced with each enzyme.
"kb" stands for kilobases, or thousands of base pairs. Which lane shows the fragments produced
when the plasmid was incubated with both EcoRI and BamH1?
a.
I
b.
II
c.
III
d.
IV
e.
V
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 14/15
7. A restriction enzyme acts on the following DNA segment by cutting both strands between
adjacent thymine and cytosine nucleotides
............TCGCGA...........
..........AGCGCT...........
Which of the following pairs of sequences indicates the sticky ends that are formed?
a.
...GCGC
CGCG...
b.
...TCGC
TCGC...
c.
...T T...
d.
...GA GA...
e.
...GCGC
GCGC
8. A segment of DNA has two restriction sites–I and II. When incubated with restriction enzymes I
and II, three fragments will be formed–a, b, and c. Which of the following gels produced by
electrophoresis would represent the separation and identity of these fragments?
a.
A
b.
B
c.
C
d.
D
e.
E
http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
P 15/15