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Biotechnology Explorer

Restriction Digestion and


Analysis of Lambda DNA

Instruction Manual

Catalog Number
166-0002-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 class-
room 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 insure
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. There is a detailed section on the laboratory
set up, complete with simple procedures which contain graphic diagrams detailing the advanced
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 contains 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 orga-
nized and logical fashion. Students who engage in the science curriculum found in the Bio-
Rad 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 Digestion and Analysis KitLinks to Biotechnology
and Genome Science
This activity 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. The techniques introduced in this exercise form the
basis of gene splicing techniques, DNA fingerprinting, and forensic DNA analysis. Using
agarose gel electrophoresis, students will analyze the migration distances, examine the diges-
tion 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. Three restriction
enzymes (EcoRI, Hind III, and Pst I) are provided, and restriction digests using Lambda DNA
will be carried out by the students. Gel electrophoresis will be employed to separate the result-
ing DNA fragments and a non-toxic blue dye (Bio-Safe DNA Staining Solution) will be
used to stain the DNA fragments for visualization. Students will then dry their gels, examine
the digestion patterns, analyze the migration distances, and determine the size of the unknown
DNA fragments.
A unique feature of the curriculum provided in this kit is that it guides students through
the procedure of constructing a standard curve. Students will use the data they obtain from their
gels to plot a line and estimate the molecular weights of the DNA restriction fragments
obtained in each lane.

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
the 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 ......................................................................................................................12

Student Manual
Lesson 1 Introduction to Restriction Analysis ........................................................15
The Structure of DNA ..............................................................................16
Restriction Enzymes are Molecular Scissors ..........................................18
LabThe Digestion ................................................................................20
Review Questions ....................................................................................22
Lesson 2 Agarose Gel Electrophoresis....................................................................24
LabDNA Fragment Separation Procedure ..........................................26
Lesson 3 Data Analysis............................................................................................29

Appendix A Teachers Answer Guide ..........................................................................39


Kit Inventory Check () List
This section lists the components provided in the Restriction Analysis Kit. It also lists
required accessories. Each kit is sufficient to outfit 8 complete student workstations. All
reagents are packaged at their proper working concentration (1x) unless otherwise noted. Use
this as a check list to inventory your supplies before beginning the experiment.
Kit Components Class Kit ()
Hind III Lambda Digest Size Standard[0.2 g/l], 100 l 1 vial
Hind III Restriction Enzyme[500 units], 40 l 1 vial
Pst I Restriction Enzyme[500 units], 40 l 1 vial
EcoRI Restriction Enzyme[500 units], 40 l 1 vial
Restriction Buffer, 500 l 1 vial
Lambda DNA Uncut - [0.2 g/l], 100 l 1 vial
Sample loading dye, 250 l 1 vial
DNA staining solution (500x), 1.5 ml 1 vial
1.5 ml Micro Tubes w/caps (color coded) 60
Foam (floating) test tube racks 8
Staining trays 8

Required Accessories - Not Included in this Kit


Micropipet (1-20 l)(catalog number166-0506-EDU) 18
Pipet tips1 box of 96(catalog 223-9038-EDU) 18
Gel Electrophoresis Chamber(catalog number 170-4400-EDU) 18
Power supply(catalog number 165-5050-EDU) 12
Electrophoresis buffer
10x TBE(catalog number 161-0733-EDU)
or 50x TAE(catalog number 161-0743-EDU) 1 liter
Agarose Powder(catalog number 162-0125-EDU) 100 grams
Permanent markers 18
Millimeter ruler 18
Lab Tape or 1/2 inch masking tape 18 rolls

Optional Accessories
Gel Support Film - for drying gels
(catalog number 170-2984-EDU) 50 sheets

1
Background Lecture Topics
One of the basic tools of modern biotechnology is DNA splicing, cutting DNA and link-
ing it to other DNA molecules. The basic concept behind DNA splicing is to remove a func-
tional DNA fragmentlets say a genefrom one organism and combine it with the DNA
of another organism in order to study how the gene works. The desired result of gene splic-
ing 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 dis-
ease, 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) DNA predictably and precisely enables
biotechnologists to recombine DNA molecules. This is where the term recombinant DNA
technology comes from. The first step in gene splicing is to locate a specific gene of interest
on a chromosome. Restriction enzymes are used to cut out the targeted gene from the rest of
the chromosome. This same enzyme will then be used to cut the DNA 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 bacteriophagesviruses that infect bacteria. Any foreign DNA
encountering a restriction enzyme is digested and cut into many fragments and rendered
ineffective. Restriction enzymes are named after the bacteria from which they are isolated. For
example:
EcoRI = The first restriction enzyme isolated from Eshericia coli bacteria
Pst I = A restriction enzyme isolated from the strain Providencia stuartii
Hind III = The third restriction enzyme isolated from Heamphious influenzae bacteria
Of the hundreds of known restriction enzymes, each one recognizes a specific nucleotide
base sequence (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 forwards and backwards on opposites strands of the DNA. For example, here
is a list of enzymes and the sites where they cut (*):

EcoRI G*A-A-T-T-C
C-T-T-A-A*G
Hind III A*A-G-C-T-T
T-T-C-G-A*A
Pst I C-T-G-C-A*G
G*A-C-G-T-C

2
Lambda Phage DNA
Lambda DNA comes from a virus called bacteriophage that infects bacteria. This virus is
harmless to man and therefore, makes an excellent and safe source of DNA for us to work with.
Below is a map of some of the genes found in the Lambda genome.
Lambda DNA is approximately 48,000 base pairs 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
Lambda DNA.

Lambda Phage Genome and Some Main Genes

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.

In
te

Ly
gr b mo

si
at ac so
ch

s
Ea

La
D
io te m

of
N
ro

rly

te
n ri e

ba
re

re
in al

sy

ct ho
gu

gu
to

nt

er st
H

la

la
he

ia
ea

Ta

tio

tio

l
si
d

il

n
s


23,130 2,027 2,322 9,416 6,557 4,361

47,813 bp

The bacteriophage Lambda and the main genes in its genome.


Arrows indicate Hind III restriction sites and the numbers


represent the size in base pairs of the DNA fragments produced.

Electrophoretic Analysis of Restriction Fragments


The three dimensional structure of a restriction enzyme allows it to fit perfectly in the
groove formed by the two complementary strands of a DNA molecule. Once attached to the
DNA, a restriction enzyme will literally slide along the double helix until it recognizes a spe-
cific sequence of base pairs which signals the enzyme to stop sliding. The enzyme then
digests (chemically separates) the DNA molecule at that sitecalled a restriction site
acting like molecular scissors, making cuts at only that specific sequence of base pairs.
If a specific restriction site occurs in more than one location on a DNA molecule, a restric-
tion enzyme will make a cut at each of those sites resulting in multiple fragments. 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 frag-
ments of varying lengths. The length of each fragment will depend upon the location of restric-
tion sites on the DNA molecule.

3
When restriction enzymes are used to cut a single strand of DNA, such as Lambda DNA,
fragments of varying sizes are produced. DNA which has been cut with restriction enzymes
can be separated using a process known as agarose gel electrophoresis. The term elec-
trophoresis means to carry with electricity.
Agarose gel electrophoresis separates DNA fragments by molecular weight. DNA frag-
ments are loaded into an agarose gel slab, which is placed into a chamber filled with a con-
ductive 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 matrix of the agarose gel acts as a molecular
sieve 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 inverse-
ly proportional to its molecular weight. Over a period of time smaller fragments will travel far-
ther than larger ones. Fragments of the same size stay together and migrate in discrete bands.
An analogy 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 students would require
more time and have difficulty working their way through the maze.
The result of electrophoresis of Lambda DNA samples digested using three different
restriction enzymes is shown in the figure below. In each case the same Lambda DNA was
used. Notice that each restriction enzyme (Pst I, EcoRI, and Hind III) produces a unique
banding pattern in each lane. The relative size of fragments contained in each band can be
determined by measuring how far each band has traveled from the origin (well) and by com-
paring the migration distances to known DNA size standards. In this example, the Pst I enzyme
has produced the smallest fragment, as depicted by the farthest migration.
1 2 3 4

Fig.1. Electrophoresis of Lambda DNA digested using three different restriction enzymes. Lane
1 contains uncut Lambda DNA. Lane 2 contains Lambda DNA digested by Pst I. Lane 3 contains
Lambda DNA digested by Eco RI. Lane 4 contains Lambda DNA digested by Hind III.

4
Implementation Time Line
There are three student lessons in this restriction analysis curriculum. 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 active laboratory sessions in this curriculum include the following activities:

Day 1 Pour gels and digest DNA


Day 2 Prepare samples, load, run, and stain gels
Day 3 Destain gels and analyze data

Lesson 1 Introduction to Restriction Analysis


Lecture and discussion
Prelab Consideration 1
Pour gels and digest samples

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 list-
ed below. It is up to the discretion of the instructor as to whether students should access com-
mon buffer solutions and equipment, or whether the teacher should aliquot solutions and
operate equipment. To avoid the potential for contamination and spills, instructors may choose
to aliquot stock solutions of DNA and enzymes to the students themselves. All other reagents
should be kept at the front of the room for student teams to access as they need them.
Number/Station ()
Lesson 1
Student workstations
Agarose gel electrophoresis system
(gel box, gel tray, 8-well comb) 1
Lab Tape 1 roll
Permanent marker 1
Instructors workstation
Molten Agarose0.8% 30 ml
Three restriction enzymes
(Hind III, EcoRI and Pst I) 1 vial each
Lambda DNA 1 vial
Restriction buffer 1 vial
Lesson 2
Student workstations
Electrophoresis Power Supply 1
Micropipet1-20 l 1
Empty microtubes (1.5 ml; 4 colors) 1
Styrofoam test tube rack 1
Permanent marker 1
Bio-Safe DNA staining solution (1x) 60 ml
Gel staining tray 1
Instructors workstation
Sample loading dye 1 vial
Electrophoresis buffer
(1x TBE or 1x TAE) 275 ml
Lesson 3
Student workstations
Water 500 ml
Millimeter ruler 1
Semi log graph paper 1
Gel support film 1 sheet
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 Introduction to Restriction Analysis


Advance Preparation
Objectives Aliquot restriction enzymes, buffers, and lambda DNA (optional)
Set up student and instructor workstations
Pour agarose gels, or, if you have your students pour their own gels dur-
ing the lab, prepare the agarose ahead of time. Agarose, when prepared
may be kept in a water bath set at 3545 C until used by the students.
Set temperature of 37 C for water bath.
Time Required Thirty minutes to 1 hour (will vary depending on how you choose to
prepare agarose gels)
Whats required Electrophoresis Gel Boxes, casting trays, and combs
Electrophoresis Buffer (TAE or TBE)
Agarose powder
40 microtubes (if you choose to aliquot enzymes, buffers, and lambda DNA)

Procedures
1. Aliquot solutions. Each student workstation will need samples of the restriction enzymes,
lambda DNA, restriction buffer, and loading dye. You may aliquot tubes for each work-
station, or you may leave the stock tubes at the front of the class for student groups to
access.
a. Aliquot 5 l of each enzyme into eight clear microtubes (total of 24 tubes). Label
the tubes: Hind III, Pst I, and Eco RI. (Notethe enzymes are very temperature
sensitive. Keep on ice at all times.) Store in the freezer until used. The day of the
lab, place the tubes on ice and distribute one tube to each team.
b. Aliquot 60 l of restriction buffer into 8 clear microtubes. Label the tubes RB.
Place tubes on ice and distribute one tube to each team.
c. Aliquot 25 l of lambda DNA into 8 clear microtubes. Label the tubes lambda.
Place tubes on ice and distribute one tube to each team.
2. Agarose preparation. The recommended gel and concentration for this classroom appli-
cation is 0.8% High Strength Analytical Grade Agarose. The recommended thickness for
the gel is 0.751.0 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.
3. Buffer preparation. TBE Electrophoresis Buffer is available as a 10x concentrated solu-
tion. TAE is available as a 50x stock concentrate. In addition to the 1x buffer needed to
make the agarose gel, approximately 275 ml is required for each electrophoresis cham-
ber. Three liters of 1x buffer will be sufficient to run eight electrophoresis chambers and
pour eight agarose gels.

7
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 concentrate, add 60 ml of concentrate to
2.94 liters of distilled water.
Use this table as a guide for gel volume requirements when casting single or multiple gels.
0.75 1.0
Gel Size cm thick cm thick
7 x 7 cm 30 ml 40 ml
4. Prepare molten agarose. Add the agarose powder to a suitable container (e.g., 250 ml
Erlenmeyer flask, Wheaton bottle, etc.). Add the appropriate amount of 1x electrophore-
sis buffer and swirl to suspend the agarose powder in the buffer. If using an Erlenmeyer
flask, you may 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 cast-
ing 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 medi-
um 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 dis-
solved. With the small flask still in place, set aside to cool to 60 C before pouring gels.
5. Pour the agarose gels. This lab activity requires that each gel has eight 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 0.50.75 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


Note: This section outlines the Tape-the-Tray method for casting gels. Simpler methods,
such as the use of casting gates, are outlined 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.

8
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 1x TAE elec-
trophoresis 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).
6. Allow the gel to solidify at room temperature for 20 minutesit 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 toward 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
Whats 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 l of loading dye into eight clear micro-
tubes. 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 sized flask. Cover the flask and store at room
temperature until ready to use. Each student team will need ~ 60 ml 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 elec-
trophoresis 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 Part 1 in Student section.

9
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 for-
mation is common.
6. Place the lid on the DNA cell carefully. Do not disturb the samples. The Sub-Cell systems
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.
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.

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 must be removed from their gel trays
in order to be placed in the staining solution. This is easily accomplished using a nonmetal-
lic 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 1015 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
Drying the Gel and Analysis of Results
Dry the Agarose Gel as a Permanent Record of the Experiment
Note: Drying agarose gels requires the use of Bio-Rads 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 is the preferred method and requires the use of Bio-Rads exclusive Gel
Support Film (catalog number 166-0199-EDU). Remove the destained agarose gel from its
staining tray and trim away any empty wells with a knife or razorblade. Place the gel direct-
ly 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 23 days. The result
will be a flat, transparent, and durable record of the experiment.

Gel Support Film

Method 1 Method 2
Method 2. After staining and destaining the gel, leave the gel in the plastic staining tray.
Let it air dry for 23 days. As the gel dries, it will shrink considerably, but proportionately.
If left undisturbed in the tray, the gel will wrinkle slightly as it dries; however, the dried gel
can be flattened and taped into a notebook for a permanent record.
To obtain a permanent record of the gel before it is dried, either trace the gel outline,
including wells and DNA bands on a piece of paper or acetate, or take a photograph using
standard cameras and film (Bio-Rads Standard Polaroid Gel Documentation System).

Graphing the Data


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 demon-
strate 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 paper in this instance. A math
extension here can also provide a perfect opportunity to explore linear and exponential
(arithmetic and geometric) sequences of numbers.

11
Quick GuideRestriction Digestion and Analysis

Sample Preparation
1. Obtain the microtubes that contain the enzyme
stock solution, Lambda DNA, and restriction
buffer. Keep all the stock solutions on ice.

DNA RB Ice

2. Obtain one of each colored microtube and


label each as follows:
clear, L = Lambda DNA
violet, P = Pst I digest
green, E = EcoRI digest L P E H
orange, H = Hind III digest

3. Pipet the reagents into each tube according to


the table below (Use a fresh tip for each trans-
fer):
tube DNA buffer Pst I EcoRI Hind III
L 4 l 6 l _ _ _
P 4 l 5 l 1 l _ _
E 4 l 5 l _ 1 l _
H 4 l 5 l _ _ 1 l

4. Mix the components by gently flicking the


tube with your finger. (If a microcentrifuge is
available, pulse spin in the centrifuge to col-
lect all the liquid to the bottom of the tube.)
Centrifuge

5. Place the tubes in the floating rack and incu-


bate for 30 minutes at 37 C.

6. After the incubation, place the samples in the


refrigerator until the next laboratory period.

Water bath

12
Agarose Gel Electrophoresis
1. Remove the digested DNA samples from the
refrigerator. Pulse spin the tubes in the cen-
trifuge to bring all of the liquid to the bot-
tom of the tube.

2. Add 2 l 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 electro-
phoresis 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. +

5. Load 10 l of each sample into separate


wells in the gel chamber in the following
order:

Lane 1: L, clear
Lane 2: P, violet
Lane 3: E, green
Lane 4: H, orange

6. Carefully place the lid on the electrophoresis


chamber. 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 1
box. Carefully remove the gel and tray from
the gel box. Be careful the gel is very slip-
pery! 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 2
gel stain overnight.

3. Pour the Bio-Safe DNA stain into a bottle


(the DNA stain can be reused). Add 60 ml
of distilled water to the gel. Let the gel
destain for 1015 minutes. Pour off the water
into a waste beaker, and analyze the data. 3

4. To obtain a permanent record, let the gel air


dry on the tray or on a piece of Gel Support
Film. When the gel is dry, tape it into your
lab notebook.
13
Student Manual

Restriction Digestion and Analysis of DNA

14
Lesson 1 Introduction to Restriction Analysis
Part 1 Background Molecular Biology
One of the basic tools of modern biotechnology is DNA splicing, cutting DNA and link-
ing it to other DNA molecules. The basic concept behind DNA splicing is to remove a func-
tional DNA fragmentlets say a genefrom 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 non-
functional 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. When 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 suc-
cessfully cut out of the chromosome. One of the ways they identify the gene they are looking
for is by its size.
Your tasks
To cut lambda DNA into a series of fragments using restriction enzymes.
To separate and sort a large group of DNA molecules by their size using agarose gel
electrophoresis.
To determine the size of each molecule separated.
You will be provided with a quantity of DNA and three different restriction enzymes.
This process is fundamental to a variety of genetic engineering techniques, including gene
splicing, DNA sequencing, gene location, forensic DNA matching, or DNA fingerprinting.
Before you begin, it might be helpful to review the structure of DNA and the activity of restric-
tion enzymes.

15
Lesson 1 Introduction to Restriction Analysis
Part 2 The Structure of DNA

Consideration How Can DNA be Cut 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 the figure below 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:

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 see in 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.

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?

16
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.

17
Lesson 1 Introduction to Restriction Analysis
Part 3 Restriction EnzymesMolecular Scissors
A major enemy of bacteria are viruses called bacteriophages. These viruses infect bacte-
ria 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. These enzymes search the viral DNA
looking for specific sequences of base pairs, a palindrome (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 1 fragment 2

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 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 spe-
cific 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.
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?

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?

19
Lesson 1 Lab
The Digestion
The DNA you will be provided with has been extracted from a bacteriophagea bac-
terium invading virus. It is known as lambda DNA (often written as: DNA.). You will be
working with three different restriction (endonucleases) enzymes. These are referred to as
Pst I, Eco RI, and Hind III.
Set up your restriction digest reaction:
1. Label four microtubes L, P, E, and H and place them in the styrofoam microtube rack.

L = No restriction enzyme - just uncut Lamba DNA


P = Pst I restriction digest of Lambda DNA
E = EcoR I restriction digest of Lambda DNA
H = Hind III restriction digest of Lambda DNA

L P E H

Describe the appearance of the dissolved DNA.

Is the DNA visible?

2. You will set up your digests in small tubes. To each tube, add 4 l of Uncut Lambda
DNA, 5 l of restriction buffer and 1 l of enzyme. We have 3 restriction enzymes from
which to choose: Pst I, EcoRI, and Hind III. Add only one kind of enzyme to a tube.
Important note: First add DNA, then restriction buffer, and then the enzymes to the
tubes. Use a fresh pipette tip for restriction buffer and each enzyme.
Fill in this chart as you go.
Lambda Rest.
Tube DNA Buffer Pst I EcoRI Hind III
P 4 l 5 l 5 l
E
H
L

20
In which tube do you expect no changes to occurthat is, no fragments produced.

What is missing in that tube that leads you to that decision?

3. Securely fasten the cap on each tube. In order to mix all reagents, hold the top of a micro-
tube between the index finger and thumb of one hand and flick 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 into the microcentrifuge, being sure to space them evenly
around the inside. Have your teacher check before spinning the tubes. Pulse spin the tubes
(hold the button for a few seconds).

Centrifuge

4. Place the sample tubes in a 37 C water bath for approximately 30 minutes or let them sit
at room temperature overnight. This process is called incubation. Restriction enzymes
work faster at the warmer temperature of 37 C.

Water bath

Note: While you are waiting, this a good time to cast your agarose gel (unless they have
already been prepared for you). Check with your teacher for the proper procedure.

21
Lesson 1 Review
Lets summarize what we learned so far
The base sequence in one strand of DNA can have a palindrome in the other strand
(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.

Apply what you have learned


A whole DNA molecule is shown below. The DNA is represented by one dark line (but
in actuality DNA has two strands).
If a DNA molecule had the restriction site A and restriction site B for a specific palin-
drome, how many fragments would be produced if it is cut by a restriction enzyme that
recognizes this palindrome?

AA B
B

Number each fragment.

Which fragment would be the largest?

Which fragment would be the smallest?

22
Draw a DNA molecule that has five randomly spaced restriction sites for a specific palin-
drome. 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.

23
Lesson 2 Agarose Gel ElectrophoresisA Molecular Strainer
ConsiderationHow 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 con-
taining 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 toward positive (+) charges.
Imagine the gel as a strainerwith 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 them-
selves. Label each fragment with its cor-
responding letter.

Have your teacher check your diagram before you proceed.

Where would the larger fragmentsthose with the greater number of base pairsbe
located; toward the top of the gel or the bottom? Why?

If 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?

24
Complete this rule for the movement of DNA fragments through an agarose gel. The
larger the DNA fragment, the

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.

In

Ly
te

si
gr b os

s
Ea
at ac om

La
D

of
ch

io te e

N
rly

te
A

ba
n ria
ro

re

re
sy
in l
m

ct o
gu

gu
to

nt

er st
H

la

la
he

ia
ea

h
Ta

tio

tio

l
si
d

il

n
s


23,130 2,027 2,322 9,416 6,557 4,361

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.

25
Lesson 2 Lab
DNA Fragment Separation Procedure

Part 1 Prepare Your Samples for Electrophoresis


ConsiderationHow Can the Pieces of DNA be Separated From One Another?
To separate the various DNA fragments from each other, the technique of gel elec-
trophoresis will be used. In order to observe 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"
to the DNA sample. 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.
1. Following incubation, obtain your four microtubes L, P, E, and H and place them in the
styrofoam microtube rack at your lab desk.
L = No restriction enzyme - just uncut Lamba DNA
P = Pst I restriction digest of Lambda DNA
E = EcoRI restriction digest of Lambda DNA
H = Hind III restriction digest of Lambda DNA
1. Dial the digital micropipet to 2.0 l and transfer this amount of loading dye to each of
the tubes marked L, P, E, and H in the microtube holder. Use a fresh tip with each sam-
ple to avoid contamination.

2. The DNA samples and the sample loading dye must be thoroughly mixed in each tube
before placing the samples in the gel wells for electrophoresis. This is easily accom-
plished by holding the top of a microtube between the index finger and thumb of one
hand and flicking the bottom of the tube gently with the index finger of the other hand.

If you have access to a microcentrifuge, place the four tubes from your microtube hold-
er (these now have DNA and loading dye) into the microcentrifuge, being sure to space
them evenly around the inside. Have your teacher check before spinning the tubes. Pulse
spin the tubes (hold the button for a few seconds). This forces all of the components to the
bottom of the tube.

26
Part 2 Set Up your Gel Electrophoresis Chamber
1. Obtain an agarose gel from your teacher, or, if your teacher instructs you to do so, pour
your own gel.
2. Place the casting tray, with the solidified gel in it, onto the central platform in the gel box.
The wells should be at the negative (-) cathode end of the box where the black electrical
lead is connected. Very carefully remove the comb from the gel by pulling it straight up.
3. Pour about 275 ml of electrophoresis buffer into the electrophoresis chamber. Pour enough
buffer into the box until it just covers the wells of the gel by 12 millimeters.

-
+

Part 3 Load your Samples and Run the Electrophoresis


1. Pipette 10 l from each tube (L, P, E, and H) into separate wells in the gel chamber. Use
a fresh tip for each tube. Gels are read from left to right. To keep things straight, the first
sample is typically loaded in the well at the upper left hand corner of the gel. For example
Lane 1 2 3 4
Sample L P E H

2. Slide the cover of the chamber into place, and connect electrical leads to the power sup-
ply, anode to anode (red to red) and cathode to cathode (black-black). Make sure both
electrical leads are attached to the same channel of the power supply.
3. Electrophorese at 120 volts for 3040 minutes. Shortly after the current is applied, the load-
ing dye can be seen moving through the gel toward the positive side of the gel chamber.
4. When electrophoresis is complete, turn off the power supply, disconnect the leads from
inputs, remove the top of gel chamber.
5. Remove the casting tray from gel chamber. The gel is very slippery. Hold the tray level.
6. Pour the excess buffer back into the original container. (It's recyclable.)

27
Part 4 Stain the DNA in Your Gel
ConsiderationHow 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 elec-
trophoresis, the DNA in the gel must be placed in a staining solution in order to visual-
ize the bands.
1. Locate the plastic staining tray at your lab station. Mark staining trays with initials and
class period.
2. Carefully place the gel into this tray by very carefully sliding it off the end of the gel tray.

3. Add enough diluted DNA staining solution to just cover the gel. That's about 60 milliliters
per tray. Cover the tray with Saran Wrap. Allow your gel to soak in the staining solution
overnight.

28
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.

Part 1 Destain Your Gel


1. Pour the DNA staining solution in your staining tray back into the container; it is recy-
clable. 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 1020 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 is allowed sit 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 mark-
er, 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 14.
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. Save the dried gel; it is part
of your experimental data.

29
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

30
Part 2 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, or a DNA standard. One of the most common DNA stan-
dards 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 dis-
tance 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 stan-
dard 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 sizes.

31
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.

32
Part 3 Analysis of DNA Fragments
The diagram you produced represents the relative position of the bands of fragments pro-
duced 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, Left Lane Right Lane
Lane
Left Lane Right
that each band moved from the well.
Measure from the bottom edge of the 1 1
1
well to the bottom edge of the band.
Record the data in the table to the right. 22 22
Label each number mm.
3
3

44

33
The number of base pairs in each of the fragments on your gel can be determined in anoth-
er 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 fragment size can be determined.

How many base pairs is fragment 2?

How accurate is this estimation of size?

34
Part 4 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 were from 2,000 to 48,000 base-
pairs, then an appropriate scale might be:
First cycle 1001,000 base pairs
Second cycle 1,00010,000 base pairs
Third cycle 10,000100,000 base pairs
When you have your vertical axis set up check with your teacher.
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.
A. For each fragment, line up a ruler vertically from the distance traveled position on the
X axis to the line that you constructed.
B. 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.

35
Semi-log Graph Paper

10,000
8,000

6,000

4,000

2,000
Size (base pairs)

1,000
800

600

400

200

100
0 5 10 15 20 25 30 35

Distance (mm)

36
Construct your own table below to record the size of each unknown fragment as deter-
mined 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.

37
Remember that the three samples of DNA started out the same. Next, each sample was
cut into pieces by the addition of three different restriction enzymes.
What evidence do you have that each enzyme cuts the DNA at different locations?

When this this activity has been completed, describe what you have done in no more than
two sentences.

Compare the two methodsdirect gel examination and semi-log graphof determining
the fragment size (in base pair units). Which method seems to be more precise? Explain
your answer.

38
Appendix A Teachers Answer Guide
Lesson 1 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 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 size fragments will be produced

39
The Digestion
Describe the appearance of the dissolved DNA.
The DNA is a colorless solution.
Is the DNA visible?
No. DNA in solution is not visible.
Fill in this chart below as you go:

Tube Lambda Buffer Pst I EcoRI Hind III


P 4 l 5 l 1 l
E 4 l 5 l 1 l
H 4 l 5 l 1 l
L 4 l 5 l

Important note: First add DNA, then restriction buffer, and then the enzymes to the
tubes. Use a fresh pipette tip for restriction buffer and each enzyme.
In which tube do you expect no changes to occurthat is, no DNA fragments produced?
The lambda tube, which gets no enzyme, should not produce any fragments.
What is missing in that tube that leads you to that decision?
No restriction enzymes are added to that tube, thus no digestion will occur and no
fragments will be produced.
This tube (L) is the control tube.
Compare tube P to tube L; What do you expect to happen in the P tube compared to the
L tube.
The P tube contains the restriction enzyme Pst I. There should be an enzymatic
digestion that occurs in that tube which results in the production of restriction frag-
ments. The L tube only contains DNA and no enzyme and so should not produce
any restriction fragments.
How can you account for what you expect to happen?
Restriction enzymes digest DNA at specific sites. If lambda DNA contains restriction
sites for the enzyme Pst I, then the DNA should be cut into fragments. With no added
enzyme, no digestion should occur.
If, at the conclusion of the reaction, the DNA in tube L becomes fragmented, what can you
conclude?
Most likely a restriction enzyme was inadvertently added to the L tube. This could
have happened by inadvertently adding an enzyme to the tube, or by not changing
pipet tips, which could result in enzyme being carried over between tubes.
Is there any visible change to the DNA after adding restriction enzymes?
No. The DNA still appears to be colorless.

40
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 5 randomly spaced restriction sites for a specific palin-
drome. 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.

41
Consideration 2 How Can you Determine if the Restriction Enzyme Works?

A piece of DNA is cut into 4four frag-


ments as shown in the diagram. A solu-
tion of the four fragments is placed in a
well in an agarose gel. Using the infor-
mation given above, draw (to the right)
how you think the fragments might sep-
arate themselves. Label each fragment
with its corresponding letter.

Have your teacher check your diagram before you proceed.

Where would the larger fragmentsthose with the greater number of base pairsbe
located; toward the top of the gel or the bottom? Why?
The large fragments would be toward 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.

42
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.

In

Ly
te

si
gr b os

s
Ea
at ac om

La
D

of
ch

io te e

N
rly

te
A

ba
n ria
ro

re

re
sy
in l
m

ct ho
gu

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23,130 2,027 2,322 9,416 6,557 4,361

47,813 bp

How many fragments were


produced by the restriction
enzyme Hind III?
6

On the gel diagram at the right,


show how you believe these frag-
ments will sort out during elec-
trophoresis.

Label each fragment with its


correct number of base pairs.

43
Consideration 4 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 Attach the dried gel showing
replica of the band patterns from the band patterns from the DNA
the DNA electrophoresis below. electrophoresis below.

44
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.

45
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.
Well
Label each band in the right lane with
6,000
its base-pair size.
5,000
Compare the two columns of bands.
Estimate the size of the fragments in
3,000
the left lane.
Upper band 5,000 1,000
Lower band 4,000
Band
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. Right Lane
Left
Left Lane
Lane Right Lane
Measure from the bottom edge of the
well to the bottom edge of the band. 11 9 11 6

Record the data in the table to the right.


22 20 22 9
Label each number in mm (millimeters).

33 20

44 29

46
The fragments of unknown length were also plotted. By placing a straight edge from the
unknown fragment toward the base pairs (bp) the fragment size can be determined.
How many base pairs is fragment 2?
~ 4000 bp
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.

47
Sample Standard Curve

Restriction Analysis Standard Curve

100,000

60,000
40,000

20,000

10,000

6,000
4,000
Size (bp)

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-axisthis is the approximate size of the fragment (C). Band 2
of Pst I is approximately 4,500 bp. Repeat this procedure for all unknown fragments in the
linear range.

48
(parenthetic values are from the data from the gel comparison of bands)

What evidence do you have that each enzyme cuts the DNA at different locations?
Each enzyme produces very different size restriction fragments that migrate with
very different patterns. This would suggest that the different enzymes are cutting at
different locations on the lambda DNA.
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 methodsdirect gel examination and semi-log graphof determining
the fragment size (in base pair units). Which method seems to be more accurate? Explain
your answer.
Both methods have their 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 Eco R1 lane, which resides between standard band
points. It is harder to estimate these intermediate sizes directly on the gel.

49
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