NAME: ________________________________
Cutting DNA Using a Restriction Enzyme
PURPOSE
To learn how to set up digestions with
restriction enzymes in order to analyze
DNA for fingerprinting identification.
INTRODUCTION
As you know, the DNA molecule can be
compared with a zipper which can be
opened up to allow replication and
transcription. Scientist have found that
there are several bacterial proteins
called enzymes, or, more specifically,
restriction enzymes, that have the
ability to cut both strands of the DNA
molecule at one specific site in the
nucleotide sequence. This is similar to
cutting a zipper into pieces by cutting
only between certain teeth of the
zipper. As a result, there are several
DNA fragments of different sizes or
different length pieces of the zipper.
These DNA fragments can then be
analyzed and use in numerous
applications of DNA technology.
MATERIALS
Latex gloves
Microfuge tube Rack
Safety Glasses
Microfuge tubes of Suspect DNA
Restriction Enzyme
Microfuge tube Crime Scene DNA
Universal Restriction Enzyme Buffer 0.510 μL Micropipette
Sterile Water
20-200 μL Micropipette
Acetylated Bovine Serum (BSA) 1:10
Micropipette Tips
Empty microfuge tubes
Permanent Marker
Container for Tip Disposal
STANDARDS ADDRESSED
Standard 1: Science As Inquiry
Benchmark 1:
The student will demonstrate the abilities
necessary to do scientific inquiry.
Indicator 3:
The student correctly uses the
appropriate technological tools and
mathematics in their own scientific
investigations.
Standard 3: Life Science
The student will develop an
understanding of the cell, molecular
basis of heredity, biological evolution,
interdependence of organisms, matter,
energy, and organization in living
systems, and the behavior of organisms.
Benchmark 1:
The student will demonstrate an
understanding of the structure and
function of the cell
Indicator 2:
The student understands cell
functions involve specific chemical
reactions.
Indicator 3:
The student understands cells
function and replicate as a result of
information stored in
deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) molecules.
Benchmark 2:
The student will demonstrate an
understanding of chromosomes,
genes, and the molecular basis of
heredity.
Indicator 1:
The student understands living
organisms contain DNA or RNA as
their genetic material, which
provides the instructions that specify
the characteristics of organisms.
Procedure
1. Before beginning, be sure to glove your
hands and put on safety glasses.
USEFUL TIP: all the reagents for this experiment
have different colors; this will aid you during the
pipetting process. Remember to check your
micropipette tip and to make sure you are
pipetting the right volume.
2. In a microfuge tube rack, place the
following stock microfuge tubes:





Questions over Procedure
Why might it be important to
have gloves while working
with DNA samples?
Diagram and label a gel box.
DNA samples (suspect DNA and crime
scene DNA)
Sterile water
Restriction Enzyme
Universal Buffer
BSA 1:10 dilution
3. Using a permanent marker, label 5 new microfuge tubes as follows:
suspect A, suspect B, suspect C, suspect D, and crime scene. These
will be referred to as the reaction tubes. Reaction tubes are necessary
because the reaction will not be carried out in the stock tubes.
4. Using the 20-200 μL micropipette place What is the purpose of this
26 μL Sterile Water into each of the
step?
reaction tubes. In order to prevent cross
contamination eject the pipette tip into
the waste container and place a new tip
on the micropipettor between each
sample.
5. Using the 0.5-10 μL micropipette place
Why is a universal buffer
4 μL Universal Buffer into each of the
used?
reaction tubes. Remember to change
tips between each sample.
6. Using the 0.5-10 μL micropipette place
What is the purpose of BSA?
4 μL BSA 1:10 into each sample DNA
tube. Remember to change tips between
each sample.
7. Using the 0.5-10 μL micropipette place
Why is it important to change
1 μL of each stock DNA sample into its tips between each sample?
corresponding reaction tube.
Remember to change tips between each
sample.
8. Using the 0.5-10 μL micropipette place
What is the purpose of adding
5 μL Restriction Enzyme into each DNA enzymes at the very end?
tube. Remember to change tips between
each sample. When you set up digestions
with restriction enzyme make sure you
always add the enzyme at the very end.
9. Gently! Tap flick each of the reaction tubes using the tip of your finger
for 20 seconds; this will allow all the solution to be mixed. Do not shake
the microfuge tubes!
10. Allow all the DNA samples to incubate
Why must the DNA samples
overnight on the lab bench at room
incubate overnight?
temperature for further analysis using
Agarose Gel Electrophoresis.
What temperature are the
DNA samples usually
incubated?
QUESTIONS AND CONCLUSIONS
1.
Name some measures that you took to prevent contamination of your DNA
samples during this lab.
2.
What is DNA fingerprinting?
3.
Propose other situations where DNA fingerprinting can be useful.
REFERENCES
1. Glencoe Biology, The dynamics of life
2. Sambroock, Molecular Biology
3. Modern Biology