Biotechnology Explorer™
Restriction Analysis of
Lambda DNA Kit
Instruction Manual
Catalog Number
166-0001-EDU
www.bio-rad.com
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
A Complete Teaching Guide
Developed over five years, Biotechnology Explorer kits and curricula have been written
for teachers, by teachers, and have been extensively field-tested in a broad range of classroom settings from high school through the undergraduate level. Easy-to-use Biotechnology
Explorer kits are the perfect way to bring the excitement of biotechnology into the classroom.
Each kit contains an innovative step by step protocol, which makes them the perfect choice
for both expert and beginning teachers.
The curriculum contained within the manual for each kit makes our products unique.
Each kit contains its own unique curriculum package which is divided into a Teachers Guide
and Student Manual. The Teachers Guide is divided into three sections which help to ensure
that the labs run smoothly. One section contains background information, lecture topics, and
suggested references which will enable each teacher, both experienced and newcomers to
biotechnology, to prepare and design lectures and lessons which can precede the actual labs.
This advance preparation will virtually insure that the labs run smoothly and that the students
will understand the concepts behind each lab.
The manuals also contain a detailed section on the laboratory set up, complete with simple procedures which contain graphic diagrams detailing the advance preparation for the labs.
In addition, this section contains time tables which will help you plan your schedule. Each lab
can be performed in a 50 minute period, which should fit into most schedules. Finally, we
provide a detailed Teachers Answer Guide which contain answers to all of the questions posed
in the Student Manual. The teacher can use these answers as a guide when reviewing or grading the questions presented in the student section of the manual.
Each kit is designed to maximize student involvement in both the labs and the thought
questions embedded in the manual. Student involvement in this process results in an increased
understanding of the scientific process and the value of proceeding into a task in an organized and logical fashion. Students who engage in the science curriculum found in the BioRad explorer kits develop a positive sense of their ability to understand the scientific method.
We strive to continually improve our curriculum and products. Your input is extremely
important to us. Incorporation of your ideas, comments, critiques, and suggestions will enable
the Explorer products to evolve into even better teaching aids.
You can find the catalog and curriculum on the internet. Look up our home page at
www.bio-rad.com or call us at 1-800-424-6723.
Ron Mardigian
Director, Biotechnology Explorer Program
ron_mardigian@bio-rad.com
Restriction Analysis—Links to Biotechnology and Genome
Science
The techniques introduced in this exercise form the basis of gene splicing techniques,
DNA fingerprinting, and forensic DNA analysis.
This kit introduces students to some important principles of genetic engineering.
Specifically, the functions of restriction enzymes and their uses as molecular biology tools
when working with DNA will be stressed. Using agarose gel electrophoresis, students will analyze the migration distances, examine the digestion patterns, and determine the sizes of the
unknown DNA fragments.
Hundreds of restriction enzymes are now known, and they have provided the catalyst for
the molecular biology revolution in the last part of the twentieth century. The restriction
enzymes studied in this investigation are Eco RI, Pst I, and Hind III. In this investigation, the
enzymes have been used to digest bacteriophage Lambda DNA. Gel electrophoresis will be
employed to separate the resulting DNA fragments and a non-toxic blue dye (Bio-Safe™ DNA
staining solution) will be used to stain the DNA fragments for visualization.
Introduction to Guided Investigation
The intent of this curriculum is to guide students through the thought process involved in
a laboratory-based scientific procedure. The focus here is not so much on the answer or result,
but rather how the result was obtained and how it can be substantiated by careful observation
and analysis of data. This is referred to as a “guided inquiry-based laboratory investigation.”
At each step along the way, student understanding of the process and the analysis of data
is stressed. Instead of providing students with explanations or interpretations, the manual poses
a series of questions to focus and stimulate thinking about all aspects of the investigation.
Table of Contents
Teachers Guide
Page
Kit Inventory Check List ..................................................................................................1
Background Lecture Topics ..............................................................................................2
Implementation Time Line................................................................................................5
Workstation Check List ....................................................................................................6
Instructors Advance Preparation ......................................................................................7
Quick Guide ....................................................................................................................13
Student Manual
Introduction ....................................................................................................................16
Lesson 1
Restriction Enzymes—Molecular Scissors............................................17
Lesson 2
Agarose Gel Electrophoresis ..................................................................21
Lesson 3
Data Analysis..........................................................................................26
Appendices
Appendix A
Teachers Answer Guide..........................................................................36
Kit Inventory Check (✔) List
This section lists the components provided in this Restriction Analysis Kit. It also lists
required accessories. Each kit is sufficient to outfit 8 complete student workstations. Use this
as a check list to inventory your supplies before beginning the experiment.
Components Included in this Kit
Class Kit
(✔)
Hind III lambda digest [0.2 µg/µl], 100 µl
Pst I lambda digest [0.2 µg/µl], 100 µl
Eco RI lambda digest [0.2 µg/µl], 100 µl
Lambda DNA Uncut [0.2 µg/µl], 100 µl
Sample Loading Buffer, 250 µl
DNA staining solution, 500x, 1.0 ml
Empty 1.5 ml Micro Tubes w/caps
(6 colors—10 each)
Foam Test Tube Racks
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
❑
60 tubes
8
❑
1–8
1–8
1–8
1–2
❑
1 Liter
1
1–8
1–8
1–8 rolls
1
❑
1 pack
❑
Required Accessories - Not Included in this Kit
Micropipet (1-20 µl), catalog number 166-0506-EDU
Pipet tips—1 box of 96, catalog number 223-9038-EDU
Electrophoresis Chamber, catalog number 170-4400-EDU
Power supply, catalog number 165-5050-EDU
Electrophoresis Buffer
10x TBE, catalog number 161-0733-EDU
or 50x TAE, catalog number 161-0743-EDU
Agarose, catalog number 162-0125-EDU
Permanent markers
Millimeter ruler
Lab Tape or 1/2 inch masking tape
Microcentrifuge (catalog number 166-0503-EDU)
Gel Support Film (50 sheets)—optional
catalog number 170-2984-EDU
1
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
Background Lecture Topics
One of the basic tools of modern biotechnology is DNA splicing, cutting DNA and linking it to other DNA molecules. The basic concept behind DNA splicing is to remove a functional DNA fragment—let’s say a gene—from one organism and combine it with the DNA
of another organism in order to study how the gene works. The desired result of gene splicing is for the recipient organism to carry out the genetic instructions provided by its newly
acquired gene. For example, certain plants can be given the genes for resistance to pests or disease, and in a few cases to date, functional genes can be given to people with non-functional
or mutated genes, such as in a genetic disease like cystic fibrosis.
This activity may be used to simulate the real world application of gene splicing. You
may suggest to your students that the DNA they are working with represents a chromosome
which will be cut into fragments. Of the fragments that are produced, one particular fragment
may represent a specific gene. This imaginary gene can code for any number of traits. But
before it can be given to a recipient organism, your students must first identify the gene by its
size using agarose gel electrophoresis.
Restriction Enzymes
The ability to cut (restrict) and paste (ligate) a functional piece of DNA predictably and
precisely is what enables biotechnologists to recombine DNA molecules. This is termed
recombinant DNA technology. The first step in DNA splicing is to locate a specific gene of
interest on a chromosome. Restriction enzymes are then used to cut out the targeted gene
from the rest of the chromosome. This same enzyme will also be used to cut the DNA of the
recipient into which the fragment will be inserted.
Restriction enzymes are biomolecules which cut (restrict) DNA at specific sites. They
were first identified and isolated in bacteria which use them as a natural defense mechanism
to cut up the invading DNA of bacteriophages—viruses that infect bacteria. Any foreign DNA
encountering a restriction enzyme will be “digested” and cut into many fragments and rendered
ineffective. These enzymes in bacteria make up the first biological immune system. Restriction
enzymes are named after the bacteria from which they are isolated. For example:
Eco RI = The 1st restriction enzyme isolated form Eshericia coli bacteria
Hind III = The 3rd restriction enzyme isolated from Heamphious influenzae bacteria
Pst I = A restriction enzyme isolated from the strain Providencia stuartii
Of the hundreds of known restriction enzymes, each one recognizes a specific nucleotide
base sequence in the DNA (restriction site) and cuts the DNA molecule at only that specific
sequence. Most restriction enzymes leave a so called sticky-end at the site where they cut
which is a short length of unpaired nucleotide bases. In general, restriction sites are palindromic, meaning they run the same sequence of bases forwards and backwards on the opposite DNA strand.
For example, here is a list of enzymes and the sites where they cut (*):
Eco RI
G*A-A-T-T-C
C-T-T-A-A*G
A*A-G-C-T-T
T-T-C-G-A*A
C-T-G-C-A*G
G*A-C-G-T-C
Hind III
Pst I
2
Lambda Phage DNA
Lambda DNA comes from a virus called bacteriophage that infects bacteria. This virus is
harmless to humans and therefore, makes an excellent and safe source of DNA for us to work
with in the laboratory. Below is a map of some of the genes found in the Lambda chromosome.
Lambda DNA is approximately 48,000 base pairs (bp) long. Since Lambda is a virus, it
is only able to express its genetic information and multiply by taking over the bacterial cell
that it infects. In this investigation, you will observe the effects of three restriction enzymes
on the DNA of the Lambda chromosome.
Lambda Phage Genome
This diagram represents a piece of DNA cut with Hind III at
each of the restrictions sites pointed to by the arrows. The
numbers represent the number of base-pairs in each fragment.
➜
6,557
➜
➜
➜
➜
l
ia
er st
ct ho
ba
of
➜
s
si
Ly
n
tio
la
gu
re
s
si
he
nt
sy
9,416
te
La
2,322
n
tio
la
gu
re
2,027
A
N
D
rly
Ea
to
in al
n ri e
io te m
at ac so
gr b mo
te
ro
In
ch
il
Ta
d
ea
H
23,130
4,361
47,813 bp
➜
The bacteriophage Lambda and the main genes in its genome.
Arrows
indicate Hind III restriction sites and the values
between the arrows represent the size in base pairs of the DNA
fragments produced.
Electrophoretic Analysis of Restriction Fragments
The three dimensional structure or shape of a restriction enzyme allows it to fit perfectly in the groove formed by the two strands of a DNA molecule. When attached to the DNA,
the enzyme slides along the double helix until it recognizes a specific sequence of base pairs
which “signals” the enzyme to stop sliding. The enzyme then digests (chemically separates)
the DNA molecule at that site—called a “restriction site”—acting like molecular scissors,
making cuts at the specific sequence of base pairs that it recognizes.
If a specific restriction site occurs in more than one location on a DNA molecule, a restriction enzyme will make a cut at each of those sites resulting in multiple fragments of DNA.
Therefore, if a given piece of DNA (linear) is cut with a restriction enzyme whose specific
recognition code is found at five different locations on the DNA molecule, the result will be
six fragments of different lengths. The length of each fragment will depend upon the location
of restriction sites on the DNA molecule.
3
A DNA molecule which has been cut with restriction enzymes can be separated using a
process known as agarose gel electrophoresis. The term electrophoresis means to carry with
electricity. Agarose gel electrophoresis separates DNA fragments by size. DNA fragments
are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive
liquid buffer solution. A direct current is passed between wire electrodes at each end of the
chamber.
DNA fragments are negatively charged, and when placed in an electric field will be drawn
toward the positive pole (the anode). The matrix of the agarose gel acts as a molecular sieve
(a matrix of holes) through which smaller DNA fragments can move more easily than larger
ones. Therefore, the distance and rate at which DNA fragments migrate through the gel is
inversely proportional to its size (in base pairs). Over a period of time smaller fragments will
travel farther than larger ones. Fragments of the same size stay together and migrate in single
“bands” of DNA. These bands will be seen in the gel after the DNA is stained.
An analogy (similar situation) would be to equate this situation to your classroom in
which all the desks have been randomly pushed together. An individual student can wind
his/her way through the chair maze quickly and with little difficulty, whereas a string of four
students would require more time and have difficulty working their way through the maze of
chairs.
The result of electrophoresis of the Lambda DNA samples digested using three different
restriction enzymes is shown in Figure 1. In each case, the same Lambda DNA was used.
Notice that each restriction enzyme (Sample 1, Uncut DNA; Sample 2, Pst I digest; Sample 3,
Eco RI digest; Sample 4, Hind III digest) produces a unique banding pattern in each lane.
The relative size of fragments contained in each band can be determined by how far each
band has traveled from the origin (well).
1
3
2
4
Fig. 1. Electrophoresis of Lambda DNA digested using three different restriction enzymes.
4
Implementation Time Line
All lessons are designed to be carried out in 50 minute periods. All lessons include:
•
A series of prelab considerations for students
•
An active student investigation
•
Questions for analysis and interpretation of results
Student Schedule
The “wet lab” portions of this investigation include the following activities:
Day 1
Day 2
Day 3
Pour gels
Load, run, and stain gels
Destain gels and analyze data
Lesson 1
Introduction to Restriction Analysis
Lecture and discussion
Prelab consideration 1
Pour gels
Lesson 2
Agarose Electrophoresis
Prelab consideration 2
Load and run gels; stain gels overnight
Lesson 3
Analysis of Results
Destain gels
Do analysis questions
Generate standard curve
Discuss results
5
Workstation Check List
Student Workstations. Materials and supplies that should be present at each student
workstation prior to beginning each lab experiment are listed below. The components provided
in this kit are sufficient for eight student workstations
Instructors (Common) Workstation. A list of materials, supplies, and equipment that
should be present at a common location that can be accessed by all student groups is also listed below. It is up to the discretion of the teacher as to whether students should access common buffer solutions/equipment, or whether the teacher should aliquot solutions and operate
equipment.
Number/Station
(✔)
Lesson 1 Introduction to Restriction Analysis
Student workstations
Agarose gel electrophoresis system
(gel box, gel tray, 8-well comb)
Lab tape
Permanent marker
1
1 roll
1
❏
❏
❏
Instructors workstation
Molten agarose—0.8%
30 ml/gel
❏
Lesson 2
Student workstation
Electrophoresis power supply
Micropipet, 1–20 µl
Empty microtubes (1.5 ml; 4 colors)
Styrofoam test tube rack
Permanent marker
Bio-Safe DNA staining solution (1x)
Gel staining tray
1
1
1
1
1
60 ml
1
❏
❏
❏
❏
❏
❏
❏
Instructors workstation
Four stock DNA samples
Uncut Lambda DNA (L)
Hind III digest (H)
Eco RI digest (E)
Pst I digest (P)
Sample loading dye
Electrophoresis buffer (1x-TBE or 1x-TAE)
1 vial
1 vial
1 vial
1 vial
1 vial
500 ml
❏
❏
❏
❏
❏
❏
500 ml
1
1
1 sheet
❏
❏
❏
❏
Lesson 3
Student workstation
Water
Millimeter ruler
Semi log graph paper
Gel support film
Instructors workstation
None required
6
Instructors Advance Preparation
This section describes preparation which may be performed in advance by the instructor.
These procedures may be carried out 1 to 2 days ahead of time by the teacher or done during
class by the individual student teams.
Lesson 1 Restriction Enzymes—Molecular Scissors
Advance Preparation
Objectives
Set up student and instructor workstations
Pour agarose gels, or, if you have your students pour their own gels
during the lab, simply prepare the agarose ahead of time. Agarose, when
prepared, may be kept in a water bath set at 35–45 °C until used.
Time required
Thirty minutes to 1 hour (will vary depending on how you choose to
prepare agarose gels)
What's required
Electrophoresis gel boxes, casting trays, and combs
Electrophoresis buffer (TAE or TBE)
Agarose powder
Procedures
Prepare Agarose Gels
1. Agarose preparation. The recommended gel and concentration for this classroom application is 0.8% High Strength Analytical Grade Agarose. The recommended thickness for
the gel is 0.75–1.0 centimeter. This concentration of agarose provides excellent resolution
and minimizes run time required for electrophoretic separation of DNA fragments. To
make a 0.8% solution, simply add 0.8 grams of agarose to 100 ml of 1x TBE or 1x TAE
electrophoresis buffer. The agarose must be made using electrophoresis buffer, not water.
2. Buffer preparation. TBE Electrophoresis Buffer is available as a 10x concentrated solution. TAE is available in 50x stock concentrate. In addition to the 1x buffer needed to
make the agarose gel, approximately 275 ml is required for each electrophoresis chamber. Three liters of 1x buffer will be sufficient to run eight electrophoresis chambers and
pour eight agarose gels. To make 3 liters of 1x TBE from a 10x TBE concentrate add
300 ml of concentrate to 2.7 liters of distilled water. To make 3 liters of 1x TAE from a
50x TAE concentrate, add 60 ml of concentrate to 2.94 liters of water.
Use this table as a guide for gel volume requirements when casting single or multiple
gels.
Gel Size
7 x 7 cm
0.75
cm thick
30 ml
7
1.0
cm thick
40 ml
3. Add the agarose powder to a suitable container (e.g., 250 ml Erlenmeyer flask, Wheaton
bottle, etc.). Add the appropriate amount of 1x electrophoresis buffer and swirl to suspend
the agarose powder in the buffer. If using an Erlenmeyer flask, invert a 25 ml Erlenmeyer
flask into the open end of the 250 ml Erlenmeyer flask containing the agarose. The small
flask acts as a reflux chamber, allowing long or vigorous boiling without much evaporation. The agarose can be melted for gel casting by boiling on a magnetic hot plate or in a
microwave oven. Heat the mixture to boiling using a microwave oven or hot water bath
until the agarose powder has melted completely.
Caution: Always wear protective gloves, goggles, and lab coat while preparing and casting agarose gels. Boiling molten agarose or the vessels containing hot agarose can cause
severe burns if allowed to contact skin.
Microwave Oven Method
Place the gel solution into the microwave. Loosen the cap if you use a bottle. Use a medium setting and set to 5 minutes. Stop the microwave oven every 45 seconds and swirl the
flask to suspend any undissolved agarose. This technique is the fastest and safest way to dissolve agarose. Boil and swirl the solution until all of the small translucent agarose particles are
dissolved. With the small flask still in place, set aside to cool to 60 °C before pouring.
Magnetic Hot Plate Method
Add a stir bar to the undissolved agarose solution. Heat the solution to boiling while stirring on a magnetic hot plate. Bubbles or foam should disrupt before rising to the neck of the
flask. Boil the solution until all of the small translucent agarose particles are dissolved. With
the small flask still in place, set aside to cool to 60 °C before pouring gels.
Pouring Agarose Gels
This lab activity requires that each gel has 8 wells, or more. Follow the instructions above
to prepare the agarose and to determine what volume of 0.8% agarose will be needed for your
class(es). Pour enough agarose to cover the gel comb teeth or to a depth of 0.5–0.75 cm. Do
not move or handle the gel tray until the gel has solidified. When solidified, gels can be stored
in zip lock bags at room temperature or in the refrigerator until use on the next day. Have
students label their plastic bags. The time needed to pour gels by an entire class is approximately 30 minutes. If possible, pour one or two extra gels for back-up.
Procedure for Casting Gels
This section outlines the Tape-the-Tray method for casting gels. Other methods are
detailed in the Sub-Cell® GT instruction manual.
1. Seal the ends of the gel tray securely with strips of standard laboratory tape. Press the
tape firmly to the edges of the gel tray to form a fluid-tight seal.
2. Level the gel tray on a leveling table or workbench using the leveling bubble provided with
the instrument.
3. Prepare the desired concentration and amount of agarose in 1x TBE or TAE electrophoresis buffer.
4. Cool the agarose to at least 60 ˚C before pouring.
5. While the agarose is cooling to 60 ˚C, place the comb into the appropriate slot of the gel
tray. Gel combs should be placed within 3/4 of an inch of the end of the gel casting tray
(not in the middle of the gel).
8
6. Allow the gel to solidify at room temperature for 10 to 20 minutes—it will appear cloudy
(or opaque) when ready to use.
7. Carefully remove the comb from the solidified gel.
8. Remove the tape from the edges of the gel tray.
9. Place the tray onto the leveled DNA electrophoresis cell so that the sample wells are at
the cathode (black) end of the base. DNA samples will migrate towards the anode (red)
end of the base during electrophoresis.
Lesson 2 Agarose Gel Electrophoresis
Advance Preparation
Objectives
Aliquot DNA loading dye (optional)
Prepare 1x Bio-Safe DNA staining solution
Set up student and instructor workstations
Time required
Thirty minutes
What's required
Electrophoresis gel boxes, casting trays, and combs
Electrophoresis buffer (TAE or TBE)
Stock solution—DNA loading dye
Stock solution—Bio-Safe DNA staining solution
Procedures
1. Aliquot loading dye (optional). Aliquot 30 µl of loading dye into eight clear microtubes.
Place tubes on ice and distribute one tube to each team.
2. Prepare Bio-Safe DNA staining solution.
Dilute the 1 ml volume of 500x DNA stain in 499 ml distilled water in an appropriate
size flask. Cover the flask and store at room temperature until ready to use. Each student
team will need ~ 60 ml of stain.
Prepare the Electrophoresis Chamber
When the agarose gel has solidified, sample loading and electrophoresis can begin.
1. When placing the gel tray into the base, make sure that the sample wells are at the cathode (black electrode) end of the base. DNA samples will migrate toward the anode (red)
end of the base during electrophoresis.
2. Prepare the required volume of 1x electrophoresis buffer (the buffer used for electrophoresis should be identical to the type used for gel preparation).
3. Submerge the gel under about 2 mm of 1x electrophoresis buffer.
4. Prepare samples for gel loading. See Lab Protocol in Student section.
5. Load the samples into the wells using standard small range micropipets.
Note: Sample wells are often difficult to see. Visualization of the well can be enhanced
by placing black paper or tape under the base or trays where comb placement or well formation is common.
6. Place the lid on the DNA cell carefully. Do not disturb the samples. The Sub-Cell system’s
lid attaches to the base in only one configuration. To attach the lid correctly, match the red
and black banana jacks on the lid with the red and black banana plugs of the base.
9
7. Power requirements vary depending on gel thickness, length and concentration, and type
of electrophoresis buffer used. For this exercise we recommend using constant voltage
(120 V) for the duration of the run.
Visualization of DNA Fragments
Bio-Safe DNA Staining Procedure
Gels should be removed from the gel tray for staining. Gel staining should be done in
individual staining trays, one per lab station. The gels need to be removed from their gel trays
in order to be placed in the staining solution. This is easily accomplished using a nonmetallic spatula. Special attention must be given to supporting the well portion of the gel since it
will crack along the well line. Pour enough stain into the tray to cover the gel(s) completely.
Stain the gels overnight in 1x Bio-Safe DNA staining solution. The next day, rinse the
stained gel with distilled water several times to reduce the background staining in the gel—
then let the gels sit in water for 10–15 minutes. To produce maximum contrast, the gels can
be destained overnight. This stain is non-toxic, however, you should use latex/vinyl gloves
while handling gels to keep your hands from being stained.
10
Lesson 3 Analysis of Results
Drying the Gel
Dry the Agarose Gel as a Permanent Record of the Experiment
Note: Drying agarose gels requires the use of Bio-Rad’s specially formulated High
Strength Analytical Grade Agarose. Other gel media may not be sufficiently formulated
for this purpose.
There are two methods that can be used to dry destained agarose gels.
Method 1
Method 1 is the preferred method and requires the use of Bio-Rad’s exclusive Gel
Support Film (catalog number 166-0199-EDU). Simply remove the destained agarose gel
from its staining tray and trim away any empty wells with a knife or razorblade. Place the
gel directly upon a piece of Gel Support Film. Center the gel on the film and let it air dry
until completely dry. As the gel dries it will bond to the film and will not shrink. If left undisturbed on the support film, the gel will dry completely at room temperature after 2–3 days. The
result will be a flat, transparent and durable record of the experiment.
Gel Support Film
Method 2
After staining and destaining the gel, leave the gel in the plastic staining tray. Let it air dry
for 2–3 days. As the gel dries it will shrink considerably, but proportionately. If left undisturbed
in the tray, the gel should remain relatively flat but will wrinkle as it dries.
11
Graphing the Data
To obtain a record of the gel before it is dried, one can either trace the gel outline, wells,
and DNA bands on a piece of paper or clear acetate, or take a photograph using standard
cameras and film (e.g., Bio-Rad Standard Polaroid Gel Documentation System).
Many of your students may not be familiar with logarithms and Semi-log graph paper. It
is suggested that you prepare a short lesson presented on the overhead or computer to demonstrate the proper way to label the coordinates and plot the points. You might also want to
include the advantage of using semi-log vs. standard graph in this instance. A math extension here can also provide a perfect opportunity to explore linear and exponential (arithmetic
and geometric) sequences of numbers.
12
Quick Guide (DNA Analysis Level 1)
Sample Preparation
1.
Obtain one of each colored microtube and
label each as follows:
clear,
violet,
green,
orange,
L = Lambda DNA
P = Pst I digest
E = EcoRI digest
H = Hind III digest
2.
Pipet 10 µl of sample from the stock tubes
and transfer to the corresponding colored
microtubes. (Use a fresh tip for each sample).
3.
You have two options:
Option one: Put the samples into the refrigerator and run the agarose gel the next day.
L
P
E
H
Option two: Prepare the samples and run
the agarose gel. (Read the agarose gel
electrophoresis quick guide.)
Agarose Gel Electrophoresis
1.
Remove the digested DNA samples from
the refrigerator. Pulse spin the tubes in the
centrifuge to bring all of the liquid to the
bottom of the tube.
Centrifuge
2.
Add 2 µl of sample loading dye into each
tube. Mix the contents by flicking the tube
with your finger.
3.
Remove the agarose gel from the refrigerator and remove the saran wrap. Fill the electrophoresis chamber and cover the gel with
1x buffer (this will require about 275 ml of
buffer).
+
4.
Check that the wells of the agarose gels are
near the black, (-) electrode and the base of
the gel is near the red, (+) electrode.
13
5.
Load 10 µl of each sample into separate wells
in the gel chamber in the following order:
Lane 1:
Lane 2:
Lane 3:
Lane 4:
6.
L, clear
P, violet
E, green
H, orange
Place the lid on the electrophoresis chamber
carefully. Connect the electrical leads into the
power supply, red to red and black to black.
+
7.
Turn on the power and run the gel at 120 V for
35 minutes.
Staining and Destaining
1.
When the electrophoresis is complete, turn off
the power and remove the top of the gel box.
Carefully remove the gel and tray from the gel
box. Be careful—the gel is very slippery. Slide
the gel into the staining tray.
2.
Add 60 ml of Bio-Safe DNA stain to the tray.
Cover the tray with Saran Wrap. Let the gel
stain overnight.
3.
Pour the DNA stain into a bottle (the Bio-Safe
DNA stain can be reused). Add 60 ml of distilled water to the gel. Let the gel destain for
10–15 minutes. Pour off the water into a waste
beaker, and analyze the data.
4.
To obtain a permanent record, simply let the
gel air dry on the tray or on a Gel Support
Sheet. When the gel is dry, tape it into your
lab notebook.
14
Student Manual
Restriction Analysis of Lambda DNA
15
Introduction
How Can Pieces of DNA Solve a Puzzle?
One of the basic tools of modern biotechnology is DNA splicing, cutting DNA and linking it to other DNA molecules. The basic concept behind DNA splicing is to remove a functional DNA fragment—let’s say a gene—from the chromosome of one organism and combine
it with the DNA of another organism in order to study how the gene works. The desired result
of gene splicing is for the recipient organism to carry out the genetic instructions provided by
its newly acquired gene. For example, certain plants can be given the genes for resistance to
pests or disease, and in a few cases to date, functional genes can be given to people with nonfunctional or mutated genes, such as in a genetic disease like cystic fibrosis.
In this lab, the DNA you will be working with is a chromosome from a virus which has
been cut into pieces with enzymes. Your task will be to determine the size of the DNA pieces
by performing a procedure known as gel electrophoresis. This involves separating a mixture
of the molecules by the size of the pieces. Once this is accomplished, you will compare your
pieces to DNA pieces of known sizes.
Of the DNA fragments that are produced, imagine that one piece in particular represents
a specific gene. This gene can code for any number of traits. But before it can be given to a
recipient organism, you must first identify the gene by using agarose gel electrophoresis.
Scientists look at the DNA in a gel to determine whether the gene they are looking for was successfully cut out of the chromosome. One of the ways they identify the gene they are looking
for is by its size.
Your tasks
To separate and sort a large group of DNA molecules by size.
To determine the size of each molecule separated.
Before you begin, it might be helpful to review the structure of DNA and the activity of
restriction enzymes.
16
Lesson 1 Restriction Enzymes—Molecular Scissors
Consideration 1 How Does DNA Become Fragmented Into Pieces?
DNA consists of a series of nitrogen base molecules held together by weak hydrogen
bonds. These base pairs are in turn bonded to a sugar and phosphate backbone. The four different nitrogen bases are Adenine, Thymine, Guanine, and Cytosine (A, T, G, and C).
Remember the base-paring rule is A = T and G = C. Refer to Figure 1 to review the structure
of a DNA molecule. If a segment of DNA is diagrammed without the sugars and phosphates,
the base-pair sequence might appear as:
Fig. 1.
Read to the right ——> A C T C C G T A G A A T T C ——->
<——-T G A G G C A T C T T A A G <—— Read to the left
Look at the linear sequence of bases (As, Ts, etc.) on each of the strands.
•
Describe any pattern you might see in the upper sequence of bases.
•
Compare the bases in the upper portion of the molecule to those in the lower portion.
Can you discover any relationship between the upper and lower strands? Describe it.
•
Now look at the upper sequence of bases and compare it to the lower. Do you notice any
grouping of bases that when read to the right and read to the left are exactly the same
order?
17
You may have discovered that the base sequence seems to be arranged randomly and that
the two strands seem to complement each other; As are paired with Ts, etc. You may have also
noticed that a portion of the top strand GAATTC (read to the right) has a counterpart in the
lower strand CTTAAG (read to the left). Similar sequences are AAGCTT and TTCGAA,
and CTGCAG and GACGTC. These sequences, called palindromes, are quite common along
the DNA molecule.
A major enemy of bacteria are viruses called bacteriophages. These viruses infect bacteria by injecting their own DNA into bacteria in an attempt to take over the operations of the
bacterial cell. Bacteria have responded by evolving a natural defense (called restriction
enzymes) to cut up and destroy the invading DNA. This could be called a primitive biological immune system. These enzymes search the viral DNA looking for certain palindromes
(GAATTCs, for example) and cut up the DNA into pieces at these sites. The actual place in
the palindrome where the DNA is cut is called a restriction site.
Look at the DNA sequence below.
Palindrome
Restriction site
fragment 2
fragment 1
A restriction enzyme cut the DNA between the G and the A in a GAATTC palindrome.
•
How many base-pairs are there to the left of the “cut”?
•
How many base-pairs are there to the right of the “cut”?
•
Counting the number of base-pairs, is the right fragment the same size as the left fragment?
•
How could you describe the fragment size in reference to the number of base pairs in the
fragment?
18
An important fact to learn about restriction enzymes is that each one only recognizes a specific palindrome and cuts the DNA only at that specific sequence of bases. A palindrome can
be repeated a number of times on a strand of DNA, and the specific restriction enzymes will
cut all those palindromes at their restriction sites, no matter what species the DNA comes
from.
•
If the GAATTC palindrome is repeated four times on the same piece of DNA, and the
restriction enzyme that recognizes that base sequence is present and digests the DNA,
how many DNA fragments will be produced?
•
If the GAATTC palindrome repeats are randomly spaced along the DNA strand, then
what can you say about the size of the fragments that will be produced?
The table below shows three of the many kind of palindromes that may be present in a
strand of DNA along with the specific enzyme that recognizes the sequence.
Name of enzyme that recognizes
the palindrome
Eco RI
Hind III
Pst I
Palindrome on the DNA molecule
GAATTC
AAGCTT
CTGCAG
Let’s summarize what we learned so far
•
The base sequence in one strand of DNA can have a palindrome in the other strand (i.e.,
GAATTC and CTTAAG).
•
Palindromes can be detected by restriction enzymes.
•
Restriction enzymes cut the palindromes at restriction sites.
•
A restriction enzyme only recognizes one specific kind of palindrome.
•
Cutting DNA at restriction sites will produce DNA fragments.
•
Fragment size can be described by the number of base-pairs they contain.
19
Applying what you have learned
A whole DNA molecule is shown below. The DNA is represented by one dark line (in
actuality, DNA has two strands).
•
If a DNA molecule had the restriction site A and restriction site B for a specific palindrome, how many fragments would be produced if it is cut by a restriction enzyme that
recognizes this palindrome?
A
A
B
B
•
Number each fragment.
•
Which fragment would be the largest?
•
Which fragment would be the smallest?
•
Draw a DNA molecule that has five randomly spaced restriction sites for a specific palindrome. How many fragments would be produced if each site were cut by a restriction
enzyme?
•
Label each fragment.
•
Rank them in order of size from largest to smallest.
AA
B
B
In this diagram A and B are different palindrome sequences on a DNA strand. Only the
restriction enzyme that recognizes site B is present.
•
Explain why only two fragments would be produced.
20
Lesson 2 Agarose Gel Electrophoresis—A Molecular Strainer
Consideration 2 How Can Fragments of DNA be Separated From One Another?
Agarose gel electrophoresis is a procedure that can be used to separate DNA fragments.
DNA is a molecule that contains many negative electrical charges. Scientists have used this
fact to design a method that can be used to separate pieces of DNA. A liquid solution containing a mixture of DNA fragments is placed in a small well formed into the gel. (The gel
looks like Jello™ dessert.) Electricity causes the molecules to move. Unlike electrical charges
attract each other; negative (-) charges move towards positive (+) charges.
Imagine the gel as a strainer with tiny pores that allow small particles to move through it
very quickly. The larger the size of the particles, however, the slower they are strained through
the gel. After a period of exposure to electricity, the fragments will sort themselves out by size.
Fragments that are the same size will tend to move together through the gel. The group will
tend to form concentrations, called bands, of pieces that are all the same size.
A piece of DNA is cut into four fragments
as shown in the diagram. A solution of
the four fragments is placed in a well in
an agarose gel. Using the information
given above, draw (to the right) how you
think the fragments might separate themselves. Label each fragment with its corresponding letter.
•
Have your teacher check your diagram before you proceed.
•
Where would the larger fragments—those with the greater number of base pairs—be
located; toward the top of the gel or the bottom? Why?
•
Suppose you had 500 pieces of each of the four fragments, how would the gel appear?
•
If it were possible to weigh each of the fragments, which one would be the heaviest?
Why?
•
Complete this rule for the movement of DNA fragments through an agarose gel.
The larger the DNA fragment, the …………
21
This diagram represents a piece of DNA cut with Hind III at
each of the restrictions sites pointed to by the arrows. The
numbers represent the number of base-pairs in each fragment.
47,813 bp
➜
The bacteriophage Lambda and the main genes in its genome.
Arrows
indicate Hind III restriction sites and the numbers
between the arrows represent the size in base pairs of the DNA
fragments produced.
•
How many fragments were
produced by the restriction
enzyme Hind III?
On the gel diagram at the right, show
how you believe these fragments will
sort out during electrophoresis.
•
Label each fragment with its
correct number of base pairs.
22
6,557
➜
9,416
➜
2,322
➜
2,027
➜
➜
➜
l
ia
er st
ct o
h
ba
of
s
n
si
tio
Ly
la
gu
re
s
te
si
La
he
nt
sy
A
N
D
n
tio
la
gu
re
rly
Ea
to
in l
n ria
io te e
at ac om
gr b os
te
m
In
ro
ch
il
Ta
d
ea
H
23,130
4,361
Lesson 2 Lab DNA Fragment Separation Procedure
Sample Preparation
1. Label four microtubes L, P, E, and H and place them in the Styrofoam microtube rack.
L
=
No restriction enzyme - just Lambda DNA
P
=
Pst I restriction digest of Lambda DNA
E
=
EcoRI restriction digest of Lambda DNA
H
=
Hind III restriction digest of Lambda DNA
(The fragment sizes are known for Hind III, thus the Lambda/Hind III is a size standard.)
L
P
E
H
2. Dial the digital micropipet to 10 µl, add a clean tip, and transfer 10 µl of Lambda DNA
cut with Pst I enzyme from the P tube (stock) on ice to the P tube in your microtube
rack. Be sure to replace the P tube (stock) back on the ice at the lab station.
3. Repeat step 2 for the P, E, and H DNA tubes that are on ice. Be sure to use 10 µl each
time: change the tip each time and place the tube (stock) back on ice at the lab station.
•
Is the DNA you added to these tubes visible?
In order to see the progress of the DNA separation on the agarose gel, we must add some
visible dye to mark or “track” the movement of these invisible DNA fragments through
the gel. This is accomplished by adding a blue dye, called a loading dye. The loading dye
actually contains two dyes; one dye that moves through the gel faster than the DNA fragments and another dye that moves slower than all the DNA fragments.
4. Re-dial the digital micropipet to 2.0 µl and transfer this amount of loading dye to each of
the tubes marked L, P, E, and H in the microtube holder. Use a new tip for all tubes.
23
5. The DNA and loading dye must be thoroughly mixed in each tube before placing the
samples in the gel wells for electrophoresis. This is easily accomplished by holding the
top of a microtube between the index finger and thumb of one hand and flicking the bottom of the tube with the index finger of the other hand. If you are using a microcentrifuge,
place the four tubes from your microtube holder (these now have DNA and loading dye)
into the microcentrifuge, being sure to space them evenly. Have your teacher check before
spinning the tubes. Pulse spin the tubes (hold the button for a few seconds). This allows
the DNA and loading dye to mix.
Centrifuge
Loading the Gel and Setting up the Gel Chamber for Electrophoresis
1. Pipette 10 µl from each tube (L, P, E, and H) into separate wells in the gel. Use a fresh tip
for each tube. Load the gel in the following order.
Lane
1
2
3
4
Tube
L
P
E
H
2. Place the lid on the electrophoresis chamber and connect the electrical leads to the power
supply, anode to anode (red to red) and cathode to cathode (black to black). Make sure
both electrical leads are attached to the same channel of the power supply.
+
3. Electrophorese at 120 volts for 35 minutes. Shortly after current is applied, the loading dye
can be seen moving through gel toward the positive side of the gel chamber.
4. When electrophoresis is complete, turn off the power supply, disconnect the leads from
the power supply inputs, and remove the top of the gel chamber.
5. Remove the gel tray from the chamber. The gel is very slippery. Hold the tray level.
6. Pour excess buffer back into the original container (it’s recyclable).
24
Consideration 3 How Can the DNA be Made Visible?
•
What color was the DNA before you added loading dye?
Since DNA is not naturally colored, it is not immediately visible in a gel. Following electrophoresis, the DNA in the gel must be placed in a staining solution in order to visualize the
bands. Remember that scientists look at the DNA in a gel to determine whether the gene they
are looking for was successfully cut out of the chromosome. One of the ways they identify the
gene they are looking for is by its size.
Staining and Destaining the Gels
Each lab station should have a small plastic staining tray.
1. Mark staining trays with initials and class period.
2. Wear rubber gloves.
3. Carefully place the gel into the tray by sliding it off the end of the gel tray.
4. Add enough DNA stain to just cover the gel.
5. Allow the gel to stain overnight.
25
Lesson 3 Data Analysis
The DNA staining solution you used in the last period has been absorbed by the agarose
gel. At this stage you will remove the excess DNA stain from the gel. Some stain will remain
in the gel itself but the DNA in the gel will be stained a dark blue color.
After destaining the gel your task will be to analyze the DNA banding patterns in the gel.
Destain Your Gel
1. Pour the DNA staining solution in your staining tray back into the container; it is recyclable. To destain your gel, rinse the gel several times with water.
2. Fill your staining tray with enough water to cover the gel. Let stand for 10–20 minutes,
then pour off the excess water. Examine DNA band patterns in each lane. Better contrast
between the DNA bands and the agarose gel will occur if the gel sits in fresh water
overnight.
3. Place your gel on a light background or a light source and record your results by making
a diagram as follows. Place a clear sheet of plastic over the gel. With a permanent marker, trace the wells and band patterns onto the plastic sheet (making a replica "picture" of
your gel). Remove the plastic sheet for later analysis.
4. Trim your gel with a knife or razor blade. Cut your gel from top to bottom to remove the
lanes that you did not load samples into, leaving only lanes 1–4.
5. Place the gel on a sheet of Gel Support Film or back into the plastic staining tray to air dry
at room temperature. The gel will stick to the film when dry. Tape the dried gel into your
laboratory notebook for a permanent record of your experiment.
26
Attach the plastic sheet that is the replica of the band patterns from the DNA
electrophoresis below.
Replica of electrophoresis gel
Attach the dried gel showing the band patterns from the DNA electrophoresis below.
Dried electrophoresis gel
27
Organize Your Data
One of the first ways of analyzing your data is to determine the approximate sizes of each
of your restriction fragments. This can be done by comparing the DNA restriction fragments
to DNA fragments of known sizes, a DNA standard. One of the most common DNA standards
is the lambda Hind III ladder.
1. Measure the distance (in millimeters) that each of your DNA fragments traveled from
the well. Measure the distance from the bottom of the loading well to the center of each
DNA band and record your numbers in the table on the next page.
2. Estimate the sizes, in base pairs, for each of your restriction fragments. Compare the distance of the unknown bands (lambda DNA, Pst I digested, and Eco RI digested) to those
of the Hind III ladder. Write the estimated sizes in the data table.
3. A more accurate way of estimating unknown DNA band sizes is to first construct a standard curve based upon the measurements obtained from the known DNA Hind III ladder.
Later in the analysis you will construct a standard curve and more accurately determine
the size of each of the DNA bands.
28
29
Data table directions. Measure the distance (in millimeters) that each fragment traveled from the
well and record it in the table. Estimate its size, in base pairs, by comparing its
position to the Hind III ladder. Remember, some lanes will have fewer than 6 fragments.
Analysis of DNA Fragments
The diagram you produced represents the relative position of the bands of fragments produced by digesting a piece of DNA of known size. This kind of digest is often called a DNA
reference ladder. Since you know the exact size and position of these fragments, they can
be used as standard reference points to estimate the size of unknown fragment bands.
Now look at the diagram of the agarose gel (below). It shows two lanes. A lane is the
column of bands below a well. The right lane contains a band pattern from four fragments of
known length (6,000, 5,000, 3,000, and 1,000 base-pairs).
•
Which lane is the reference ladder?
How do you know?
Well
•
Label each band in the right lane with
its base-pair size.
•
Compare the two columns of bands.
Estimate the size of the fragments in
the left lane.
Band
Agarose gel
Upper band
Lower band
•
How did you determine the sizes of the
two bands in the left lane?
Examine the practice gel above.
•
Measure the distance in millimeters,
that each band moved from the well.
LeftLane
lane
Left
Right Lane
lane
Right
Measure from the bottom edge of the
well to the bottom edge of the band.
1
1
11
•
Record the data in the table to the right.
22
22
•
Label each number ....... mm.
3
3
4
30
The number of base pairs in each of the fragments on your gel can be determined in another way that sometimes can be more accurate. This involves graphing the size of the known
fragments (the Hind III digest fragments) against the distance each band (fragment) moved
through the gel. This will provide you with a more precise relationship of distance of travel
through the gel with fragment size. This is most conveniently done on semi-log graph paper.
Look at the data from the practice gel above. The fragments of known size were plotted
on graph paper producing the curve shown below.
x = Known fragment size
o = Unknown fragment size
8,000 bp
7,000 bp
6,000 bp
5,000 bp
Unknown fragment 1
4,000 bp
Unknown fragment 2
3,000 bp
2,000 bp
1,000 bp
Millimeters
The fragments of unknown length were also plotted. By placing a straight edge from the
unknown fragment toward the base pairs (bp) the fragments size can be determined.
•
How many base pairs is fragment 2?
•
How accurate is this estimation of size?
31
Determining the Size of the DNA Fragments From Your Laboratory Data
Obtained by Gel Electrophoresis
From your laboratory data, you were able to estimate the approximate size of each of the
DNA fragments that you separated on your gel. This was done in terms of the number of base
pairs.
•
Explain how you made this determination.
You have been provided with a three-cycle, semi-log graph paper. This means that the
vertical axis of this paper is divided into three sections, each gradated from 1 to 10. The following steps might provide some assistance in your graphing.
1. Fragment size will be on be on the vertical (Y) axis. Each cycle of 1 to 10 should start with
a value 10 times greater than the previous cycle.
2. Since the range of base pairs in your known sample was from 2,000 to 48,000 base-pairs,
then an appropriate scale might be:
First cycle -
100–1,000 base pairs
Second cycle -
1,000–10,000 base pairs
Third cycle -
10,000–100,000 base pairs
When you have your vertical axis set up, check with your teacher before proceeding.
3. The horizontal (X) axis should have your scale for distance traveled through the gel in millimeters. This can be treated as in any other graph.
4. For your fragments of known size (use the Hind III digest), plot the distance traveled in
relationship to fragment size for each fragment. Connect as many of the points as you
can by drawing a straight line through them. This will provide a “standard curve” with
which you will be able to determine the size of your unknown fragments from the other
two samples.
To determine the size of an unknown fragment.
1. For each fragment, line up a ruler vertically from the “distance traveled” position on the
X axis to the line that you constructed.
2. From the point where your ruler intersected your line, place the ruler horizontally and
note where it intersects with the Y axis for fragment size. This will be your determination
of the size for that fragment.
32
Semi-log Graph Paper
10,000
8,000
6,000
4,000
Size (base pairs)
2,000
1,000
800
600
400
200
100
0
5
10
15
Distance (mm)
33
20
25
30
35
•
Construct your own table below to record the size of each “unknown” fragment as
determined by the semi log graphing procedure. It might also be interesting to indicate on
this same table the values you arrived at by comparing band positions in the original gel
analysis. Compare the two sets of values.
34
•
When this Data Table has been completed, describe what you have done to determine
DNA fragment sizes in this investigation. Use no more than two sentences.
•
Explain how you think you could make your DNA size estimation more accurate.
•
Compare the two methods—direct gel examination and semi-log graph—of determining
the fragment size (in base pair units). Which method seems to be more accurate?
Explain your answer.
35
Appendix A Teachers Answer Guide
Lesson 1 Restriction Enzymes Consideration Questions
Look at the linear sequence of bases (As, Ts, etc.) on each of the strands.
•
Describe any pattern you might see in the upper sequence of bases.
There is no specific type of pattern associated with the upper sequence of bases.
•
Compare the bases in the upper portion of the molecule to those in the lower portion.
Describe any relationship you can see.
A always pairs with T; G always pairs with C.
•
Now look at the upper sequence of bases and compare it to the lower. Do you notice any
grouping of bases that when read to the right and read to the left are exactly the same
order?
CTTAAG.
A restriction enzyme cut the DNA between the G and the A in a GAATTC palindrome.
•
How many base-pairs are there to the left of the cut?
4
•
How many base-pairs are there to the right of the cut?
10
•
Counting the number of base-pairs, is the right fragment the same size as the left fragment?
No, it is larger.
•
How could you describe the fragment size in reference to the number of base pairs in the
fragment.
Fragment 1 is a 4 base pair fragment.
Fragment 2 is a 10 base pair fragment.
•
If the GAATTC palindrome is repeated four times on the same piece of DNA, and the
restriction enzyme that recognizes that base sequence is present, how many DNA
fragments will be produced?
5
•
If the GAATTC palindrome repeats are randomly spaced along the DNA strand, then
what can you say about the size of the fragments that will be produced?
Random sized fragments will be produced.
36
Applying What You Have Learned
•
If a DNA molecule had the restriction sites A and B for a specific palindrome, how many
fragments would be produced?
3
•
Number each fragment.
•
Which fragment would be the largest?
Fragment 3.
•
Which fragment would be the smallest?
Fragment 2.
•
Draw a DNA molecule that has five randomly spaced restriction sites for a specific palindrome. How many fragments would be produced if they were each cut by a restriction
enzyme?
Answers will vary.
•
Label each fragment
Answers will vary.
•
Rank them in order of size from largest to smallest.
Answers will vary.
In this diagram A and B are different palindrome sequences on a DNA strand. Only the
restriction enzyme that recognizes site B is present.
•
Explain why only two fragments would be produced.
The enzyme would cut at site B, producing two DNA fragments.
37
Lesson 2 Agarose Gel Electrophoresis
Consideration 2
A piece of DNA is cut into four fragments as shown in the diagram. A solution of the four fragments is placed in a
well in an agarose gel. Using the information given above, draw (to the right)
how you think the fragments might separate themselves. Label each fragment
with its corresponding letter.
•
Have your teacher check your diagram before you proceed.
•
Where would the larger fragments—those with the greater number of base pairs—be
located; toward the top of the gel or the bottom? Why?
The large fragments would be towards the top of the gel because it is more difficult
for the larger pieces to strain through the gel.
•
Suppose you had 500 pieces of each of the four fragments, how would the gel appear?
There would still be only 4 bands present.
•
If it were possible to weigh each of the fragments, which one would be the heaviest?
Why?
Fragment D would be heaviest because it comprises the largest piece of DNA and
would thus have the greatest mass.
•
Complete this rule for the movement of DNA fragments through an agarose gel.
The larger the DNA fragment, the slower it migrates through an agarose gel.
38
This diagram represents a piece of DNA cut with Hind III at
each of the restrictions sites pointed to by the arrows. The
numbers represent the number of base-pairs in each fragment.
How many fragments were
produced by the restriction
enzyme Hind III?
6
On the gel diagram at the right,
show how you believe these fragments will sort out during electrophoresis.
•
Label each fragment with its
correct number of base pairs.
39
6,557
➜
9,416
➜
2,322
➜
➜
➜
2,027
47,813 bp
•
➜
l
ia
er st
ct ho
ba
of
s
si
on
i
Ly
at
l
gu
re
s
te
si
La
he
nt
sy
A
N
D
n
tio
la
gu
re
rly
Ea
to
in l
n ria
io te e
at ac om
gr b os
te
m
In
ro
ch
il
Ta
d
ea
H
23,130
4,361
Lesson 3 Lab
Consideration 3 How Can the DNA be Made Visible?
What color was the DNA before you added loading dye?
The DNA is a colorless solution.
Attach the plastic sheet that is the
replica of the band patterns from
the DNA electrophoresis below.
40
Attach the dried gel showing
the band patterns from the DNA
electrophoresis below.
41
Data Table Directions. Measure the distance (in millimeters) that each fragment traveled from the
well and record it in the table. Estimate its size, in base pairs, by comparing its position to the
Hind III ladder Remember: some lanes will have fewer than 6 fragments.
Analysis of Your DNA Fragments
•
Which lane is the reference ladder?
How do you know?
The right lane is the reference ladder
because it contains the known DNA
fragments.
•
Label each band in the right lane with
its base-pair size.
•
Compare the two columns of bands.
Estimate the size of the fragments in
the left lane.
Upper band
Lower band
•
6,000
5,000
3,000
5,000
4,000
1,000
How did you determine the sizes of the
two bands in the left lane?
Compared both unknown bands to
the migration of the known bands in
the reference lane.
Examine the Practice Gel Above
•
Measure the distance, in millimeters,
that each band moved from the well.
Measure from the bottom edge of the
well to the bottom edge of the band.
•
Record the data in the table to the right.
•
Label each number in mm (millimeters).
42
Right lane
Right
Lane
LeftLane
lane
Left
1
1
9
11
6
2
2
20
22
9
33
20
4
29
The fragments of unknown length were also plotted. By placing a straight edge from the
unknown fragment toward the base pairs (bp) the fragments size can be determined.
•
How many base pairs is fragment 2?
~ 4000 bp.
•
How accurate is this estimation of size?
Because this standard curve is fairly linear, this estimation is fairly accurate, probably within 10% of the true value.
From your laboratory data you were able to estimate the approximate size of each of the
DNA fragments that you separated on your gel. This was done in terms of the number of
base pairs.
•
Explain how you made this determination:
Each of the unknown fragments was compared to the migration of the closest band
in the reference lane. Because the sizes of the Hind III standard bands are known,
you could estimate the size of the unknown fragments based upon their positions
relative to the Hind III bands in the gel.
43
Sample Standard Curve
Restriction Analysis Standard Curve
100,000
60,000
40,000
20,000
Size (bp)
10,000
6,000
4,000
2,000
1,000
600
400
200
100
10
15
20
25
30
35
Distance (mm)
For example, band 2 of Pst I migrated 23.5 mm (A). From the 23.5 mm mark on the
X-axis, read up to the standard line; when you intersect your standard curve, mark the spot
with a shaded circle (B). Follow the intersect point over to the Y-axis and determine where
the graph line meets the Y-axis—this is the approximate size of the fragment (C). Band 2 of
Pst I is approximately 4500 bp. Repeat this procedure for all unknown fragments in the
linear range.
44
(parenthetic values are from the data from the gel comparison of bands)
•
When this Data Table has been completed, describe what you have done to determine
DNA fragment sizes in this investigation. Use no more than two sentences.
The first determination of size involved the approximation of unknown DNA band
size by comparison to the migration of known DNA samples directly on the agarose
gel. The second determination more accurately determined unknown DNA size by
plotting a standard curve from known DNA bands, and then using the curve to
determine the sizes of unknown samples.
•
Explain how you think you could make your DNA size estimation more accurate.
Drawing two standard curves, rather than one, would make the size estimation more
accurate. One curve could be drawn for the larger data points (bands 1, 2, and 3) and
a second curve could be drawn for the smaller points (4, 5, and 6). Estimation of
unknown fragment sizes could then be made from the most appropriate curve.
•
Compare the two methods—direct gel examination and semi-log graph—of determining
the fragment size (in base pair units). Which method seems to be more accurate? Explain
your answer.
Both methods have advantages and disadvantages. With the gel examination method,
it is possible to estimate sizes over the entire range of the gel, in particular for
extremely large fragments. Because large fragments are outside of the linear range
of the standard curve, you can not accurately estimate sizes from the curves, but
you can estimate the sizes from the gel.
Use of the semi-log graph standard curve is very accurate within the linear range.
The logarithmic cycles on the graph paper allow you to accurately estimate sizes of
fragments, such as band 3 of the EcoRI lane, which resides between standard band
points. It is harder to estimate these intermediate sizes directly on the gel.
45
Bio-Rad
Laboratories
Life Science
Group
ISO 9001
registered
www.bio-rad.com
Bio-Rad Laboratories Main Office 2000 Alfred Nobel Drive, Hercules, California 94547, Ph. (510) 741-1000, Fx. (510)741-5800
Also in: Australia Ph. 02-9914-2800, Fx. 02-9914-2889 Austria Ph. (1)-877 89 01, Fx. (1) 876 56 29 Belgium Ph. 09-385 55 11, Fx. 09-385 65 54
Canada Ph. (905) 712-2771, Fx. (905) 712-2990 China Ph. (86-10) 2046622, Fx. (86-10) 2051876 Denmark Ph. 39 17 9947, Fx. 39 27 1698
Finland Ph. 90 804 2200, Fx. 90 804 1100 France Ph. (1) 43 90 46 90, Fx. (1) 46 71 24 67 Germany Ph. 089 31884-0, Fx. 089 31884-100
Hong Kong Ph. 7893300, Fx. 7891257 India Ph. 91-11-461-0103, Fx. 91-11-461-0765 Israel Ph. 03 951 4127, Fx. 03 951 4129
Italy Ph. 02-21609.1, Fx. 02-21609.399 Japan Ph. 03-5811-6270, Fx. 03-5811-6272 The Netherlands Ph. 0313 18-540666, Fx. 0313 18-542216
New Zealand Ph. 09-443 3099, Fx. 09-443 3097 Singapore Ph. (65) 272-9877, Fx. (65) 273-4838 Spain Ph. (91) 661 70 85, Fx. (91) 661 96 98
Sweden Ph. 46 (0) 8 627 50 00, Fx. 46 (0) 8 627 54 00 Switzerland Ph. 01-809 55 55, Fx. 01-809 55 00
United Kingdom Ph. 0800 181134, Fx. 01442 259118
4006101 Rev A