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The Biotechnology Education Company ®
sed
Revi nd
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Upd
EDVO-Kit #
206
Restriction Enzyme
Mapping
Storage: See Page 3 for

specific storage instructions
EXPERIMENT OBJECTIVE:
The objective of this experiment is to develop an
understanding of DNA mapping by determining
restriction enzyme cleavage sites on a circular DNA plasmid.
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EVT 206.121217
206
Experiment
Restriction Enzyme Mapping
Table of Contents
Page
Experiment Components 3
Experiment Requirements 4
Background Information 5
Experiment Procedures
Experiment Overview and General Instructions 8
Restriction Enzyme Reactions 9
Agarose Gel Electrophoresis 11
Size Determination of DNA Restriction Fragments 13
Study Questions 16
Instructor’s Guidelines 17
Notes to the Instructor 18
Pre-Lab Preparations 20
Experiment Results and Analysis 23
Study Questions and Answers 25
Appendices 27
Material Safety Data Sheets 37
EDVOTEK, The Biotechnology Education Company, and InstaStain are registered

trademarks of EDVOTEK, Inc. UltraSpec-Agarose and FlashBlue are trademarks of
EDVOTEK, Inc.
All components are intended for educational research only. They are not to be used for
diagnostic or drug purposes, nor administered to or consumed by humans or animals.
THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA.

None of the experiment components are derived from human sources.
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2 EVT 206.121217
206 Experiment
Experiment Components
Experiment # 206
Storage
is designed for
6 groups. A HindIII endonuclease Freezer
B Bgl I endonuclease Freezer
C Restriction enzyme dilution buffer Freezer
D Restriction enzyme reaction buffer Freezer
E Water, qualified enzyme grade Freezer
F Supercoiled plasmid DNA Freezer
G Standard DNA fragments Freezer
Reagents & Supplies

This Experiment is designed
• UltraSpec-Agarose™ powder for DNA staining with
• Concentrated electrophoresis buffer InstaStain® Blue. or Flash-
• 10x Gel Loading Solution Blue™ liquid stain.
• 1 ml pipet
• Microtipped Transfer Pipets Alternatively, DNA may be
• Microtest tubes with attached caps stained with InstaStain®
• InstaStain® Blue Ethidium Bromide, which
• FlashBlue™ liquid stain must be purchased separately
(Cat. # 2001).
STORAGE OF PERISHABLES
This experiment includes perishable components (A-G) which were sent on wet ice. Store
these components at -20° C (-4° F). Please note what type of freezer you have and store
components accordingly.
Frost-free Freezer
Most refrigerator/freezers in homes are frost free. This means the freezer goes through
warming cycles to eliminate frost (defrost cycle). If using this type of freezer, keep the en-
zymes in the foam chest (with the ice brick) in which they were sent. This will help main-
tain the enzymes at -20° C when the freezer goes through the defrost cycle.
Non Frost-free Freezer

These older model freezers, which are still sold but are harder to find, do not go through
warming cycles. Therefore, ice will build up on freezer walls over time. If using this type
of freezer, check to make sure that it maintains temperature at -20° C.
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FAX: 202.370.1501 • email: info@edvotek.com
EVT 206.121217 3
206
Experiment
Requirements
• Horizontal gel electrophoresis apparatus

• D.C. power supply
• Automatic micropipets with tips
• Waterbath (37° C or 45° C)
• Balance
• Microwave, hot plate or burner
• Pipet pump
• 250 ml flasks or beakers
• Hot gloves
• Safety goggles and disposable laboratory gloves
• Distilled or deionized water
• Ice
• For gel staining with InstaStain® Blue or FlashBlue™ liquid stain
• Small plastic trays or large weigh boats for destaining
• White light DNA visualization system
• For gel staining with InstaStain® Ethidium Bromide
• UV Transilluminator
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4 EVT 206.121217
Experiment 206
Background Information
Restriction enzymes are endonucleases that cleave both strands of DNA at spe-
cific sequences of bases. The cleavage site is usually located within or near the A
recognition site. The location of restriction enzyme cleavage sites are important 2000
in analysis, mapping of genetic structure and in molecular cloning experiments.
Restriction enzyme mapping determines the relative positions of cleavage sites Enzyme
to one another in a DNA molecule. This is done by determining sizes of DNA A+C
fragments generated by different combinations of restriction enzyme digests. 3000
As an example, consider a 5000 base pair, circular plasmid DNA containing
single recognition sites for enzymes A, B, and C. Any one of these enzymes will C
cleave the DNA once to produce a linear molecule of 5000 base pairs. Differ-
Figure 1: A + C
ently paired combinations of enzymes in the same reaction mixture (double-
digests) will produce the following DNA fragments (sizes in base pairs):
A+B A+C B+C A+B+C 500

A
4500 3000 3500 3000
500 2000 1500 1500 B
500
Enzyme
1500
A+B+C
The smallest DNA fragment generated in a reaction represents the shortest distance
between two cleavage sites. Analysis of a double digest can be done by arbitrarily
C 3000
placing one of the cleavage sites at the top of a circle. This site acts as a reference
point. The closest cleavage site to this point can be placed in a clockwise or counter-
Figure 2
clockwise direction.
The data from the double digests can be put together in a pattern that is consistent
with all the observed fragment sizes that are generated by the co-digestion. The 500
A
triple digest, A + B + C (Figure 3), is a confirmatory test for constructing the map.
When one compares the digest of A + C (Figure 1) and the triple digest, the map B
places cleavage site B between A and C. The two different relative positions of B to
Enzyme
A and C are shown in Figures 2 and 3. A+B+C 1500
Generally, a restriction enzyme map is constructed by first determining the number

3000 C
of fragments each individual enzyme produces. The size and number of fragments
is determined by electrophoresis. Reactions containing various combinations of
enzymes are performed to determine distances between cleavage sites. The size of Figure 3
every fragment produced is calculated from data obtained by agarose gel electro-
phoresis. The relative positions of the cleavage sites are then ordered in a way that is
consistent with all the observed fragment sizes. Determination of site order requires
choosing one of the cleavage sites as an arbitrary starting point at 12 o’clock on a cir-
cle (position 1). Usually, this is an enzyme that has a single cleavage site in the DNA.
Using the shortest distances between sites, as determined in the double digests, the
sites are placed on a circle (or a line, depending on the DNA).
This can be visually demonstrated by a description of an example as shown in Figure

4. A circle is drawn on a transparent sheet for an overhead projector with a position
A marked. On a separate transparency, a circle of a smaller diameter is drawn with a
position B marked. Position B represents the action of the second enzyme which also
cuts the plasmid only once. This process is continued with circles of decreasing di-
ameters, which are each on separate sheets and have a single marked location. The
transparencies can be overlaid and individually turned clockwise or counter clockwise
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
Copyright © 1987-2012 EDVOTEK, Inc., all rights reserved. EVT 206.121217
5
206 Experiment
so that the relative positions predict the fragment sizes obtained from co-digests or
multiple digests.
Gel electrophoresis is a convenient analytical method for determining the size of DNA
molecules in the range of 500 to 30,000 base pairs. The agarose gel consists of micro-
scopic pores that act as a molecular sieve. The gel is placed in a chamber containing a
buffer solution and electrodes. Samples of DNA are loaded into wells made in the gel
during casting. Current is applied from a D.C. power source. Since DNA is strongly
negative at neutral pH, it will migrate through the gel towards the positive electrode.
The pores in the gel separate the DNA molecules according to their size and shape.
The migration rate of DNA molecules having the same shape is inversely proportional
to their size. This means that the smaller the DNA molecule, the faster it migrates
through the gel.
The charge to mass ratio is the same for different sized DNA molecules. The reason for
this is inherent in the structure of the molecule. The bases in DNA are linked together
by negatively charged phosphodiester bonds. For every base pair (average molecular
weight of approximately 660) there are two charged phosphate groups. Therefore,
the net charge is accompanied by approximately the same mass. The absolute amount
of charge on the molecule is not a critical factor in the separation process. The separa-
tion occurs because smaller molecules pass through the pores of the gel more easily
than larger ones, i.e., the gel primarily separates linear pieces of DNA based on physical
size.
DNA fragments of unknown sizes are determined with the use of Standard DNA frag-
ments of known size. They are electrophoresed in parallel with Standard DNA frag-
ments. After electrophoresis, the DNA bands are visualized, and the migration distanc-
es of the known and unknown fragments are measured.
The standard fragments are used to make a standard curve on semi-log graph paper by
plotting their sizes on the y-axis versus the migration distance on the x-axis. The size of
the fragments on the y-axis are expressed as the log of the number of base pairs or the
log of their molecular weight.

This allows most of the data to
A
A be plotted as a straight line.
Clockwise B The migration distance of
the unknown is found on the
C x-axis and its size is estimated
B
A from the standard curve.
B
C
If a DNA molecule contains
several recognition sites for a
A restriction enzyme, then under
C B
certain experimental condi-
tions, it is possible that certain
Counter C
sites are cleaved but not
others. These incompletely
Clockwise cleaved fragments of DNA are
Figure 4
called partial digests (partials).
6
Experiment 206
Recognition Sites
Partials can arise if an insufficient amount of enzyme is used or
the reaction is stopped after a short time (Figure 5). Reactions A B C
containing partials may also contain some molecules that have
been completely cleaved. Complete Partial
Cleavage Cleavage
Circular DNAs such as plasmids are supercoiled. Supercoiled
forms of DNA have a more compact and entangled shape (like a C B C
twisted rubber band) than their corresponding non-supercoiled B A B
forms (linear, nicked and relaxed circles). When supercoiled
DNA is cleaved by a restriction enzyme once on each of the two A C
strands, it unravels to its linear form. If supercoiled DNA is
B
nicked (a phosphate bond is broken anywhere in the molecule,
in either strand) it unravels to form a nicked circle (Figure 6). A
Figure 5
Under the electrophoresis conditions used in this experiment,
supercoiled DNA migrates faster than its linear form and linear
DNA migrates faster than its nicked circular form. During
replication, several plasmid molecules can form interlocking rings with
themselves. These forms are called catenanes. Catenanes can contain 2
plasmid molecules (dimer), three molecules (trimer), etc. (Figure 7). Cat-
enanes migrate more slowly than single circular DNA that are nicked during
electrophoresis. Dimers migrate faster than trimers, which migrate faster
than tetramers, etc. Catenanes give rise to the same final restriction en-
zyme cleavage patterns as their uncatenated (single circular) forms. Partial
restriction enzyme digestion products are also possible with plasmids con- Figure 6
taining single recognition sites. In such cases, there can be some remaining
uncut supercoiled plasmid, an accumulation of nicked form (single-stranded
cut) and some linear product.
In this experiment you will determine the locations of restriction enzyme cleavage sites on a
circular plasmid DNA. Standard DNA fragment sizes are provided to construct the standard
curve. The enzymes used to cleave the plasmid are HindIII and
Bgl I. Assume the Bgl I site is at position 0. The objective is to
calculate the distances between the points of cleavage and to
determine their relative orientations. Quick Reference:
Standard DNA fragments, which were

generated by restriction enzymes are
provided in this experiment. A standard
curve will be plotted on semi-log graph
paper. The following are the Standard DNA
fragment sizes - length is expressed in base
pairs.
Dimer Trimer 23130 9416 6557

4361 3000 2322
Figure 7 2027 725 570
7
206 Experiment
Experiment Overview and General Instructions
EXPERIMENT OBJECTIVE:
The objective of this simulated forensic analysis is to develop an understanding of the
use of restriction enzymes as applied to RFLP-based DNA fingerprinting.
LABORATORY SAFETY GUIDELINES:

1. Wear gloves and goggles while working in the
laboratory.
Experiment Procedure
2. Exercise caution when working in the laboratory –

you will be using equipment that can be dangerous
if used incorrectly.
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4. Always wash hands thoroughly with soap and water after working in the labora-
tory.
5. If you are unsure of something, ASK YOUR INSTRUCTOR!
LABORATORY NOTEBOOKS:
Scientists document everything that happens during an experiment, including experi-
mental conditions, thoughts and observations while conducting the experiment, and,
of course, any data collected. Today, you’ll be documenting your experiment in a labo-
ratory notebook or on a separate worksheet.
Before starting the Experiment:

• Carefully read the introduction and the protocol. Use this information to form a
hypothesis for this experiment.
• Predict the results of your experiment.
During the Experiment:

• Record your observations.
After the Experiment:

• Interpret the results – does your data support or contradict your hypothesis?
• If you repeated this experiment, what would you change? Revise your hypothesis
to reflect this change.
8
Experiment 206
Restriction Enzyme Reactions
GENERAL INSTRUCTIONS
Quick Reference:
Add enzyme(s), reaction mixtures to the DNA to start the reactions.
Use a fresh micropipet tip to dilute the DNA or enzymes. Component Label
• Mix by tapping the tube after transfer of reagents. Plasmid DNA DNA
Restriction Enzyme
• Incubate reactions at 45° C or 37° C in a waterbath. Reaction Reaction Buffer Rxn Buffer
tubes can be suspended in a test tube rack that is partially im- Enzyme Grade water Water
mersed. Standard DNA Fragments Markers
Diluted HindIII HindIII
• Gel loading solution contains protein denaturants that stop the Diluted Bgl I Bgl I
enzyme reactions. Do not place a pipet that has been in gel
loading solution into a tube containing enzymes until you are
ready to terminate the reaction.
REACTION INSTRUCTIONS
1. Label microtest tubes 1 through 4 for four restriction enzyme digestion reactions.
Put your initials or group number on the tubes.
2. Tap all the tubes on the lab bench to collect all the contents at the bottom of the
tube.
3. Use an automatic micropipet to dispense 30 µl of Rxn Buffer (enzyme reaction buf-

fer) to each of the four reaction tubes labeled 1 through 4.
4. Add DNA, water and enzyme to reaction tubes as summarized in the table below.
Use a FRESH micropipet tip for each transfer of DNA or enzyme.
5. Cap reaction tubes and tap gently to mix. There should be no dense glycerol layer
of enzyme solution at the bottom of the reaction tube.
6. Tap each tube on the lab bench to collect the contents at the bottom.
7. Incubate the tubes in a 45° C waterbath for 15 minutes or in a 37° C waterbath

for 30-60 minutes.
Chart II: Options for

Chart I: Summary of Restriction Enzyme Reactions Restriction Enzyme
Incubation
Reaction E Reaction 10x Total
Qualified A B
Rxn Buffer DNA Hind III Bgl I Volume Gel Load Sample Waterbath Incubation
Tube (µl) Water (µl) (µl) Volume
(µl) (µl) (µl) (µl) Temperature Time
(µl)
1 30 10 10 - - 50 5 55 45° C 15 min.
2 30 10 5 5 - 50 5 55 37° C 30-60 min.
3 30 10 5 - 5 50 5 55
4 30 10 - 5 5 50 5 55
9
206 Experiment
Restriction Enzyme Reactions
After incubation is completed: Important

8. Add 5 µl of 10x gel loading solution to reaction tubes Reminder:
1 - 4 to stop the reactions. Cap and mix by tapping.
The reactions are ready for electrophoresis. Do not cross-con-
taminate enzyme and
DNA stocks by using
OPTIONAL STOPPING POINT the same pipet tip.
The prepared DNA samples can be stored in the

freezer for electrophoresis at a later time.
10
Experiment 206
Agarose Gel Electrophoresis
Prepare the Gel (see Appendices A, B, and C):

1. Prepare an agarose gel with specifications summarized below.
• Agarose gel concentration required: 0.8% Wear

Gloves & goggles
• Recommended gel size: 7 x 7 cm
• Number of sample wells required: 5
• Well-former template (comb)

placement (using EDVOTEK units): One 6-well comb in first set of notches
Load the Samples:
2. Load 40 µl of each of the DNA samples in the following manner.
Optional Step: Heat the samples, including the Standard DNA frag- Reminders:
ments for two minutes at 65ºC. Allow the samples to cool for a few
minutes. During electrophoresis,
the DNA samples migrate
Lane Tube through the agarose gel to-
1 Markers Standard DNA Fragments wards the positive electrode.
2 1 Plasmid DNA (uncut) Before loading the samples,
3 2 Plasmid DNA cut with HindIII make sure the gel is properly
4 3 Plasmid DNA cut with Bgl I oriented in the apparatus
5 4 Plasmid DNA cut with HindIII and Bgl I chamber.
– +
Black Red
Run the Gel: Sample wells
3. Close the lid of electrophoresis chamber and connect the electrodes. Set the
power source to the required voltage and run the gel for the length of time
specified by your instructor.
4. Check that the current is flowing correctly. Remember, the DNA samples will
migrate towards the positive (red) electrode during electrophoresis.
5. Turn off the power supply as soon as the gel has finished running.
11
206 Experiment
Agarose Gel Electrophoresis
Stain the Gel:

6. Carefully remove the agarose gel and casting tray from the electrophoresis cham-
ber.
7. Stain the gel according to the staining instruction (See Appendices E-H).
DO NOT STAIN GELS IN THE ELECTROPHORESIS APPARATUS!
View the Gel:

8. Examine your stained gel and document the results of your experiment.
9. This can be done by taking a picture of your gel or by tracing the gel onto a trans-
parency. Be sure to include the outline of the gel and the sample wells.
12
Experiment 206
Size Determination of DNA Restriction Fragments
The first step for mapping DNA restriction sites is to determine the migration distance
and size of the DNA fragments generated after electrophoresis. The assignment of sizes
for DNA fragments separated by agarose gel electrophoresis can have ± 10% margin of
error. The sizes of the "unknowns" (three digests in lanes 3, 4, and 5) will be extrapolat-
ed by graphing their migration distances relative to the Standard DNA Fragments (lane
1), for which the size of each fragment is known.
1. Measure and record the distance traveled in the agarose gel by each Standard DNA
fragment (except the largest 23,130 bp fragment, which will not fit in a straight line
in step 4).
In each case, measure from the lower edge of the sample well to the lower edge of
each band. Record the distance traveled in centimeters (to the nearest millimeter).
2. Label the semi-log graph paper:
• Label the non-logarithmic horizontal x-axis "Migration Distance" in centimeters

at equal intervals.
• Label the logarithmic vertical y-axis "Log base

pairs". Choose your scales so that the data 100,000
9
points are well spread out. The first cycle on 8
7
the y-axis represents 100-1,000 base pairs and 6 Example showing the
the second cycle represents 1,000-10,000 base 5 plots of marker fragment
4 migration distances on the
pairs.
3
non-logarithmic x-axis
3. For each Standard DNA fragment, plot the mea- versus its size, in base pairs,
on the logarithmic y-axis
sured migration distance on the x-axis versus its 2
size in base pairs, on the y-axis. 10,000 base pairs

4. Draw the best average straight line through all 10,000
9
the points. 8
7
Log base pairs
(logarithmic y-axis)
6
The line should have roughly equal numbers of 5
points scattered on each side of the line. Some 4
points may be right on the line (see example at left). 3
5. Measure migration distance of each of the DNA 2
fragments from the three restriction enzyme

digestions (reactions 2, 3 and 4).
1,000
9
6. Using the graph of the Standard DNA fragments, 8
7 1,000 base pairs
determine the sizes in base pairs of each fragment 6
from the restriction enzyme digestion reactions 5
• Find migration distance of unknown frag- 3
ment on x-axis - draw vertical line from that

2
point until standard graph line is intersected.
• From the point of intersection, draw a second 100

line horizontally to the y-axis and determine 1 cm 2 cm 3 cm 4 cm 5 cm
the approximate size of the fragment in base Migration Distance
pairs (refer to example at right).
13
206 Experiment
Size Determination of DNA Restriction Fragments
MAPPING RESTRICTION SITES ON PLASMID DNA
The size of the plasmid used in this experiment is 4340 bp.
1. Draw a circle representing a 4340 bp plasmid on a transparent sheet of acetate.
• Mark the positions of the HindIII sites corresponding to the sizes of the frag-
ments obtained on the gel.
2. Draw a second circle representing a 4340 bp plasmid on a transparent sheet of

acetate.
• Mark the position of the Bgl I site at the top (12:00 o'clock).
3. To draw a composite map of both HindIII and Bgl I, overlay the HindIII map on top
of the Bgl I map.
• Keeping the Bgl I site at the 12:00 o'clock position, rotate the HindIII map until
the relative distances between the sites approximate the relative sizes of the
fragments generated upon digestion.
• Specify, in base pairs, the distances between all the sites.
4. Compare fragment sizes of the single digests of HindIII and Bgl I

(Reactions 2 and 3) with fragments obtained in double digest with both HindIII and
Bgl I (Reaction 4).
Note: Because DNA is circular, clockwise and counter clockwise rotation described
in step 3 will yield identical fragments. Additional mapping (not included in this experi-
ment) is required to differentiate between the two maps.
14
100,000
90,000
80,000
70,000
60,000
50,000
40,000
30,000
20,000
10,000
9,000
8,000
7,000
6,000
5,000
Y-axis: Base Pairs
4,000
3,000
2,000
1,000
900
800
700
600
500
400
300
200
100
1 cm 2 cm 3 cm 4 cm 5 cm 6 cm
X-axis: Migration distance (cm)

206 Experiment
Study Questions
Answer the following study questions in your laboratory notebook or on a separate

worksheet.
1. Can the size of a supercoiled plasmid DNA be determined by using standard DNA
size fragment electrophoresed in parallel?
2. An unknown DNA molecule was cleaved using several restriction enzymes indi-
vidually and in various combinations. The DNA fragment sizes were determined
by agarose gel electrophoresis and the restriction enzyme recognition sites were
mapped. Subsequently, the DNA was sequenced and an extra recognition site
was found for one of the enzymes. However, all the other mapping data was
consistent, within experimental errors, with sequence data. What are the simplest
explanations for this discrepancy? Assume the DNA sequence had no errors.
3. A plasmid was cleaved with several restriction enzymes, individually and in combi-
nations. The following fragment sizes (base pairs) were determined by agarose gel
electrophoresis.
Eco RI 4363
Ava I 4363
Pvu II 4363
Pst I 4363
Eco RI - Ava I 2938 1425
Eco RI - Pst I 3609 754
Ava I - Pvu II 3722 641
Ava I - Pst I 2182 2181
Pvu II - Pst I 2820 1543
Eco RI - Pvu II 2297 2066
Make a restriction map based on this data. Note: there may be some slight dis-
crepancy in summing up the total base pairs. Indicate the distances between sites.
Why is only one band detected in the Ava I - Pst I co-digest.
16
206
Instructor’s Guide Experiment
Notes to the Instructor:

Class size, length of laboratory sessions, and availability of equipment are factors
which must be considered in the planning and the implementation of this experi-
ment with your students. These guidelines can be adapted to fit your specific set
of circumstances. If you do not find the answers to your questions in this section,
a variety of resources are continuously being added to the EDVOTEK web site.
In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time
zone. Call for help from our knowledgeable technical staff at 1-800-EDVOTEK
(1-800-338-6835).
Online Ordering NATIONAL CONTENT AND SKILL STANDARDS

now available
By performing this experiment, students will learn to perform restriction en-
zyme analysis, load samples and run agarose gel electrophoresis. Analysis of the
experiments will provide students the means to transform an abstract concept
into a concrete explanation. Please visit our website for specific content and skill
standards for various experiments.
Visit our web site for

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EDVOTEK Electrophoresis Experiments are easy to perform and are designed for
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EVT 206.121217 17
206 Experiment
This experiment simulates a forensic case in which DNA samples from a hypothetical
crime scene and suspects are digested by six-base cutting enzymes (Eco RI and HindIII).
The objective is to analyze suspect DNA fingerprint patterns and compare them with
“crime scene” samples. Each DNA sample will be cleaved with two restriction enzymes
in separate reactions, and pairs of fragmentation patterns will serve as the fingerprints.
The DNA fragmentation patterns will be analyzed in the stained agarose gel, without
the need for Southern blot analysis.
This experiment module contains biologicals and reagents for six groups. The experi-
mental procedures consist of two major parts: 1) restriction enzyme digestion of DNA,
which is followed by 2) agarose gel electrophoresis.
Each laboratory group receives two predigested, ready-for-electrophoresis “crime

scene” samples and standard DNA fragments (markers). Four additional DNA samples
Instructor’s Guide
are generated by performing restriction enzyme digestion reactions on the DNAs of

two suspects.
If you have six (6) electrophoresis units, one for each of the six lab groups, electropho-
resis can be performed simultaneously by all six groups. Alternatively, some lab groups
can store their samples at 4° C and perform the electrophoresis at different times.
APPROXIMATE TIME REQUIREMENTS

1. Prelab prep and dispensing of biologicals and reagents take approximately 1-2
hours.
2. The approximate time required for students to perform the restriction enzyme
digestion and prepare samples for electrophoresis is 50 minutes. The incubation
time for restriction enzyme digestion may be extended to 60 minutes.
3. Gel preparation: Whether you choose to prepare the gel(s) in advance or have the
students prepare their own, allow approximately 30-40 minutes for this procedure.
Generally, 20 minutes of this time is required for gel solidification.
4.
Practice Gel Loading: If your students are unfamiliar with using micropipets and
sample loading techniques, a practice activity is suggested prior to conducting
the experiment. EDVOTEK electrophoresis experiments contain a tube of practice
gel loading solution for this purpose. Casting of a separate
Table practice gel is highly recommended. This activity can require
Time and Voltage Guidelines
C.1 (0.8% Gel) anywhere from 10 minutes to an entire laboratory session,
EDVOTEK Electrophoresis Model depending upon the skill level of your students.
M6+ M12 & M36
Volts Minimum / Maximum Minimum / Maximum 5. Conducting Electrophoresis: The approximate time for electro-
phoresis will vary from approximately 15 minutes to 2 hours.
150 15 / 20 min 25 / 35 min Different models of electrophoresis units will separate DNA
125 20 / 30 min 35 / 45 min at different rates depending upon its design configuration.
Generally, the higher the voltage applied the faster the samples
70 35 / 45 min 60 / 90 min migrate. However, maximum voltage should not exceed the
50 50 / 80 min 95 / 130 min indicated recommendations. Refer to Table C.1 for specific Time
and Voltage recommendations.
18
Experiment 206
GEL STAINING AND DESTAINING AFTER ELECTROPHORESIS
This experiment features FlashBlue™ and InstaStain® Blue for gel staining after elec-
trophoresis. It is a proprietary new staining method which saves time and reduces liquid
waste. EDVOTEK also offers InstaStain® Ethidium Bromide and Protein InstaStain® for
staining protein polyacrylamide gels.
Three options are provided for gel staining with FlashBlue™ liquid and InstaStain® Blue.
• Method 1 - Staining and Visualization of DNA with FlashBlue™ liquid stain:
A simple and rapid staining procedure that will allow gels to be stained in less than
5 minutes.
• Method 2 - One-step Staining and Destaining with InstaStain® Blue:
Agarose gels can be stained and destained in one easy step, which can be completed
in approximately 3 hours, or can be left in liquid overnight.
• Method 3 - Direct Staining with InstaStain® Blue:
Using InstaStain® Blue cards requires approximately 5-10 minutes for staining. DNA
bands will become visible after destaining for approximately 20 minutes, and will
become sharper with additional destaining. For the best photographic results, allow
the gel to destain for several hours to overnight. This will allow the stained gel to
"equilibrate" in the destaining solution, resulting in dark blue DNA bands contrast-
ing against a uniformly light blue background.
19
206 Experiment
Pre-Lab Preparations
Many enzymes are labile. Keep them cold and minimize handling. Do not
dilute the enzymes into their reaction buffers more than 30 minutes before
Important the lab. Once diluted, the enzymes must be used; they can no longer be
Reminders stored. Remove enzyme stocks from the freezer (will still be liquid) and put
them directly on ice shortly before they are required for pre-lab prepara-
tions.
There should not be any dense layer of enzyme solution left at the bottom
of the stock tubes if properly mixed after dilution.
Allow ample time to equilibrate a water bath at 45° C or 37° C on the day
students perform the experiment.
PREPARATION OF GENERAL REAGENTS

Quick Reference: Components
for Restriction Enzyme Mapping 1. Thaw the restriction enzyme dilution buffer, concen-
trated reaction buffer, enzyme grade water and DNAs
(C, D, E, F & G) on ice.
A HindIII endonuclease
B Bgl I endonuclease • Tap the tubes with your fingers or on a table to get
C Restriction enzyme dilution buffer all contents to the bottom of the tube. Put them
D Restriction enzyme reaction buffer back on ice.
E Water, qualified enzyme grade
2. Component E is qualified enzyme grade water, which
F Supercoiled plasmid DNA
students will use in the restriction enzyme reactions.
G Standard DNA fragments
• Label six tubes "Water".
• Dispense 25 µl of the water to each tube.
3. Component G contains Standard DNA fragments (markers).
• Label six tubes "Markers".

• Dispense 36 µl of Standard DNA fragments to each tube.
4. Dispense 130 µl of restriction enzyme reaction buffer (D) to each of six

microtest tubes labeled "Rxn Buffer". Place tubes on ice.
PREPARATION OF PLASMID DNA

5. To prepare the Plasmid DNA, pipet to a clean microtest tube and mix by
tapping the tube:
190 µl (0.19 ml) restriction enzyme reaction buffer (D)

90 µl supercoiled plasmid DNA (F)
6. Dispense 45 µl of the plasmid DNA to each of six microtest tubes labeled

"DNA".
20
Experiment 206
PREPARATION OF RESTRICTION ENZYMES

The restriction enzyme stocks will still be liquid when removed from freezer storage.
7. Tap the tubes of enzymes with your fingers or on a table to get all the contents to
the bottom of the tube. Put them back on ice.
8. Prepare the HindIII restriction enzyme.
• With a fresh pipet tip, transfer 75 µl of ice cold restriction enzyme dilution buf-
fer (C) to the HindIII tube (A ).
• Cap the tube and tap to mix. Place the tube back on ice.
• Label six tubes “HindIII”
• Transfer 15 µl to each of the six tubes.
• Cap the tubes and place on ice.
9. Prepare the Bgl I restriction enzyme.
• With a fresh pipet tip, transfer 75 µl of ice cold restriction enzyme dilution buf-
fer (C) to the Bgl I tube (B).
• Cap the tube and tap to mix. Place the tube back on ice.
• Label six tubes “Bgl I”
• Transfer 15 µl to each of the six tubes.
• Cap the tubes and place on ice.
Restriction enzyme digestion for

Quick Reference: Components Expt. 206: each group requires:
for Restriction Enzyme Mapping
A HindIII endonuclease 130 µl Restriction Enzyme Reaction Buffer

B Bgl I endonuclease 45 µl Plasmid DNA
C Restriction enzyme dilution buffer 25 µl Enzyme Grade Water
45 µl Standard DNA Fragments
D Restriction enzyme reaction buffer
15 µl Diluted HindIII (on ice)
E Water, qualified enzyme grade 15 µl Diluted Bgl I (on ice)
F Supercoiled plasmid DNA
G Standard DNA fragments Automatic micropipet and tips
4 microtest tubes with attached caps
Marking pen
10x Gel loading solution
21
206 Experiment
QUICK REFERENCE TABLES FOR PRE-LAB PREPARATIONS
Many enzymes are labile. Keep them cold and minimize handling. Do not dilute the
enzymes into their dilution buffers more than 30 minutes before the lab. Once diluted,
the enzymes must be used; at the most, they can be stored overnight at -20° C.
Important Remove enzyme stocks from

Reminders the freezer (will still be liquid) Quick Reference: Components
and put them directly on for Restriction Enzyme Mapping
ice shortly before they are
required for pre-lab prepara- A HindIII endonuclease
tions. B Bgl I endonuclease
C Restriction enzyme dilution buffer

There should not be any D Restriction enzyme reaction buffer
dense layer of enzyme solu- E Water, qualified enzyme grade
tion left at the bottom of the F Supercoiled plasmid DNA
stock tubes if properly mixed G Standard DNA fragments
after dilution.
Quick
Reference Preparation of Biologicals & Reagents
for 6 Groups
Dispense for
Tube Component Label each tube
(µl)
Restriction Enzyme
D Rxn Buffer 130
Reaction Buffer
D+F Plasmid DNA DNA 45

Enzyme Grade Water
E Water
25
G Standard DNA Markers 45

Fragments
A+C HindIII 15
Diluted HindIII (on ice)
B+C BglI 15
Diluted BglI (on ice)
Recommended dispensing volumes include a small amount of "excess", which is

5-10 μl more than the total volume required for the experiment.
22
Experiment 206
Experiment Results and Analysis
GEL ELECTROPHORESIS RESULTS
The migration rate of DNA fragments is inversely

proportional to the log of their size in base pairs.
However, the 23,130 base pair fragment is usually
not included in the standard curve. Larger DNA
fragments have different charge:mass ratios and
tend to migrate faster than one would predict.
This is potentiated by increasing gel porosity and
electric field strength. Recommended conditions
for this experiment minimize this effect, but it is
still present.
The idealized representation of the uncut control
DNA (lane 2) shows (proceeding up the gel,
towards the negative electrode) the following:
supercoiled monomer, supercoiled dimer, and
nicked (relaxed) circular. Higher catenanes are not
shown. Upon digestion with a restriction enzyme
Note: This technique that cuts DNA once, such as Bgl I in this experi-
has a ± 10 - 15% ment, will convert all forms to the linear form as shown in lane 4. The size
margin of error. of the linear DNA is 4340 bp, as opposed to those shown in lane 2.
Representative results are shown in the idealized schematic of the gel, which
shows the relative positions of the DNA restriction fragments, but are not
depicted to scale. Actual results will yield bands of varying intensities.
Lane 1 Standard DNA Fragments

(expressed in approximate base pairs)
23,130 9,416 6,557 4,361 3,000

2,322 2,027 725 570
Lane 2 Plasmid - superhelical DNAs cannot be precisely

measured using linear standard fragments.
Lane 3 HindIII 3,710 bp ± 556

630 bp ± 95
Lane 4 Bgl I 4,340 bp ± 650
Lane 5 HindIII + Bgl I 2,080 bp ± 312

1,630 bp ± 245
630 bp ± 95
23
206 Experiment
Experiment Results and Analysis
PLASMID MAPS OF REACTIONS 1-4
Hind III
630
Hind III
Reaction 1
Reaction 2
Control Hind III
(Uncut plasmid
3710
3710 + 630 = 4340

Bgl I Bgl I
Reaction 3
Bgl I Reaction 4
2080 1630
Hind III + Bgl I
4340
Hind III
Hind III 630
1630 + 630 = 2260

4340 - 2260 = 2080
24
Please refer to the kit
insert for the Answers to
Study Questions
206
Experiment
Notes:

26 EVT 206.121217
206 Experiment
Appendices
A Individual Preparation for 0.8 % Agarose Gel Electrophoresis
B Quantity Preparations for 0.8 % Agarose Gel Electrophoresis
C Agarose Gel Preparation Step by Step Guidelines
D Loading the Samples and Conducting Electrophoresis
E Staining & Visualization of DNA with FlashBlue™ Liquid Stain
F One-step Staining and Destaining of DNA with InstaStain® Blue
G Direct Staining of DNA with InstaStain® Blue
H General Notes on Staining & Destaining

EVT 206.121217 27
206 Experiment
Appendix
Individual Preparation for 0.8% Agarose Gel Electrophoresis
(DNA Staining with FlashBlue™ or InstaStain® Blue)
A
We provide concentrated (50x) Tris-Acetate-EDTA (TAE) buffer for agarose gel electro-
phoresis. Dilute the concentrate using 49 volumes of distilled or deionized water for
every one volume of buffer concentrate. Prepare volume of buffer as required for your
electrophoresis apparatus.
Individual Gel Casting and Buffer: Each laboratory group is responsible for preparing
their own gel and buffer.
If preparing a 0.8% gel with If preparing a 0.8% gel with

concentrated (50x) buffer, use Table A.1 diluted (1x) buffer, use Table A.2
Table Table Individual 0.8%*

A.1 Individual 0.8%* UltraSpec-Agarose™ Gel A.2 UltraSpec-Agarose™ Gel
DNA Staining with FlashBlue™ or InstaStain® Blue DNA Staining with FlashBlue™
or InstaStain® Blue
Amt of Concentrated Distilled Total
Size of Gel Agarose + Buffer (50x) + Water = Volume Amt of Diluted
Size of Gel
(cm) (g) (ml) (ml) (ml) Agarose + Buffer (1x)
(cm)
(g) (ml)
7x7 0.23 0.6 29.4 30 7x7 0.23 30
7 x 10 0.39 1.0 49.0 50 7 x 10 0.39 50
7 x 14 0.46 1.2 58.8 60 7 x 14 0.46 60
* 0.77 UltraSpec-Agarose™ gel percentage rounded up to 0.8%
Table
Electrophoresis (Chamber) Buffer Prepare the agarose gel solution for a single gel in a 250 ml
B
Dilution flask or beaker.
EDVOTEK Total Volume Distilled
50x Conc.
Model # Required (ml)
Buffer (ml) + Water (ml)
Note: The UltraSpec-Agarose™ bottle usually contains exactly
M6+ 300 6 294 three grams. If the amount of agarose is not specified on the
M12 400 8 392 label or if the bottle’s plastic seal has been broken, weigh the
20
agarose to ensure that the correct amount is used.
M36 1000 980
Table
C.1 (0.8% Gel)
Time and Voltage recommendations for EDVOTEK EDVOTEK Electrophoresis Model
equipment are outlined in Table C.1 for 0.8% aga- M6+ M12 & M36
rose gels. The time for electrophoresis will vary from Volts Minimum / Maximum Minimum / Maximum
approximately 15 minutes to 2 hours depending
150 15 / 20 min 25 / 35 min
upon various factors. Conduct the electrophoresis for
the length of time determined by your instructor. 125 20 / 30 min 35 / 45 min
70 35 / 45 min 60 / 90 min
50 50 / 80 min 95 / 130 min
28
Experiment 206
Quantity Preparations
Appendix
for Agarose Gel Electrophoresis
B
To save time, the electrophoresis buffer and agarose gel solution can
be prepared in larger quantities and distributed among an entire class.
Unused diluted buffer can be used at a later time and solidified aga- Table
rose gel solution can be remelted. D Bulk Preparation of
Electrophoresis Buffer
Bulk Electrophoresis Buffer Concentrated Distilled Total
Buffer (50x) + Water = Volume
Quantity (bulk) preparation for 3 liters of 1x electrophoresis buffer is (ml) (ml) (ml)
outlined in Table D.
60 2,940 3000 (3 L)
Batch Agarose Gels (0.8%)

Table
1. Use a 500 ml flask to prepare the diluted gel buffer. Batch Preparation of
E.1 0.8% UltraSpec-Agarose™
2. Pour 3.0 grams of UltraSpec-Agarose™ into the prepared buffer.
Swirl to disperse clumps. Amt of Concentrated Distilled Total
Agarose + Buffer (50X) + Water = Volume
(g) (ml) (ml) (ml)
3. With a marking pen, indicate the level of solution volume on the
outside of the flask. 3.0 7.5 382.5 390
4. Heat the agarose solution as outlined previously for individual gel

preparation. The heating time will require adjustment due to the Note: The UltraSpec-Aga-
larger total volume of gel buffer solution. rose™ kit component is often
labeled with the amount it
5. Cool the agarose solution to 60° C with swirling to promote even contains. In many cases, the
60˚C entire contents of the bottle
dissipation of heat. If evaporation has occurred, add distilled water
to bring the solution up to the original volume as marked on the is 3.0 grams. Please read the
flask in step 3. label carefully. If the amount
of agarose is not specified or
if the bottle's plastic seal has
6. Dispense the required volume of cooled agarose solution for casting each gel.
been broken, weigh the aga-
The volume required is dependent upon the size of the gel bed and DNA staining
rose to ensure you are using
method, which will be used. Refer to Appendix A for guidelines.
the correct amount.
7. Allow the gel to completely solidify. It will become firm and cool to the touch after
approximately 20 minutes. Then proceed with preparing the gel for electrophoresis.
Preparing Gels in Advance

Solidified agarose gels can be removed from the casting trays and stored in the refrigera-
tor for up to 2 weeks before performing the experiment. Keep gels covered in buffer to
prevent them from drying out.
Gels that have been removed from their trays for storage should be “anchored” back to
the tray. Place a few drops of molten agarose on the casting tray before replacing the
gel. This will secure the gel to the tray and prevent it from sliding and/or floating in the
electrophoresis chamber.
NEVER store an agarose gel at -20° C. FREEZING WILL DESTROY THE GEL!
29
206 Experiment
Appendix
Agarose Gel Preparation - Step by Step Guidelines
C 1. Mix agarose powder with 1x electrophoresis buffer in a flask. Swirl the mixture
to disperse clumps of agarose powder. Using a lab pen, mark the level of the
solution volume on the outside of the flask.
If gel trays and rubber end The amount of agarose and buffer required depends upon the size of the cast-
caps are new, they may be initially ing tray. Refer to the Appendix A for specifics.
somewhat difficult to
assemble. Here is a helpful hint: 2. Dissolve agarose by boiling the solution until it appears completely clear.
Be sure to check the solution regularly while heating. If you see crystalline par-
ticles, the agarose is not completely dissolved. Larger total volumes of gel buffer
solution will require longer heating time.
A. Microwave method: At high

• Cover the flask with plastic wrap to minimize altitudes, use a
evaporation. microwave oven
Place one of the black end caps
with the wide “u” shaped slot fac- • Microwave the solution on High for 1 minute. BE to reach boiling
ing up on the lab bench. CAREFUL—the flask may be hot! Carefully remove temperatures.
the flask from the microwave and swirl to mix.
Push one of the corners of the gel
tray into one of the ends of the • Continue heating the solution in 15-second bursts until the agarose is
black cap. Press down on the tray completely dissolved.
at an angle, working from one end
to the other until the end of the B. Hot plate method:
tray completely fits into the black • Cover the flask with aluminum foil to minimize evaporation.
cap. Repeat the process with the • Swirling occasionally, bring the mixture to a boil over the burner, and
other end of the gel tray and the remove from heat as soon as the agarose is completely dissolved.
other black end cap.
3. Cool the agarose solution to 60° C with careful swirling to promote even dissipa-
tion of heat. The flask should now be warm to the touch, but not painfully hot
to handle.
If considerable evaporation has occurred during heating, add distilled water to

bring the solution to the original volume and swirl to mix. Placing the bottle in
a 60° C water bath, if available, will allow the agarose to cool, while preventing
it from prematurely solidifying.
4. While agarose is cooling, close off the open ends of a clean and dry casting tray
using rubber dams or tape.
A. Using rubber end caps:

Position a rubber dam at each end of the bed.
Make sure the dam makes firm contact with the
sides and bottom of the bed.
B. Using labeling or masking tape:

• Extend one-inch wide tape over the sides and
bottom edge of the tray.
• Fold the extended tape edges back onto the
sides and bottom. Apply pressure at contact
points to establish a reliable seal.
30
Experiment 206
Agarose Gel Preparation Step by Step Guidelines, continued
Appendix
5. Place the casting tray on a level surface. Position a well tem-
plate (or “comb”) into one set of notches, near the end of the C
tray. Make sure the comb is secure and rests evenly across the
tray.
6. Seal the sides of the gel tray to prevent agarose solution from
leaking.
• Using a transfer pipet, deposit a small amount of the aga-
rose to the interior edge on both sides of the tray.
• Wait approximately 1 minute for the agarose to solidify.
7. Once it has cooled to 60° C, slowly pour the agarose solution into the
tray.
DO NOT POUR BOILING HOT AGAROSE INTO THE GEL TRAY!

Extreme heat can cause the tray to warp or crack! DO NOT
POUR
8. Allow the gel to solidify completely. BOILING
HOT 60˚C
The gel will stiffen and become less transparent as it solidifies. The AGAROSE
gel should thoroughly solidify within 20 minutes. INTO THE
GEL BED.
9. Slowly and carefully remove the comb and dams. Take particular
Hot agarose solution
care with the comb to prevent damage to the wells.
may irreversibly warp
the bed.
10. At this point, the gel is ready for electrophoresis (or storage under
buffer in the refrigerator).
31
206 Experiment
Appendix
Loading the Samples and Conducting Electrophoresis
D 1. Load the samples as instructed by your instructor.
2. After the DNA samples are loaded, carefully snap the cover down onto the elec-
trode terminals.
Make sure that the negative and positive color-coded indicators on the cover and
apparatus chamber are properly oriented.
3. Insert the plug of the black wire into the black input of the power source (negative
input). Insert the plug of the red wire into the red input of the power source (posi-
tive input).
4. Set the power source at the required voltage and conduct electrophoresis for the
length of time determined by your instructor. General guidelines are presented in
Table C.1.
5. Check to see that current is flowing properly - you should see bubbles forming on
the two platinum electrodes.
6. After the electrophoresis is completed, turn off the power, unplug the power
source, disconnect the leads and remove the cover.
7. Remove the gel from the bed for staining.
About DNA Gel Staining

• After electrophoresis, the agarose gels require staining in order to visualize the
separated DNA samples. This experiment features two proprietary stain called
FlashBlue™ liquid stain and InstaStain® Blue cards.
• Check with your instructor regarding which staining method you should use.
Table
C.1 (0.8% Gel)
EDVOTEK Electrophoresis Model
M6+ M12 & M36
Volts Minimum / Maximum Minimum / Maximum
150 15 / 20 min 25 / 35 min

125 20 / 30 min 35 / 45 min
70 35 / 45 min 60 / 90 min
50 50 / 80 min 95 / 130 min
32
Experiment 206
5 Min. Staining
Staining and Visualization of DNA
Appendix
FlashBlue™ Liquid Stain
E
METHOD 1:
1. Dilute 10 ml of 10x FlashBlue™ with 90 ml of distilled or deionized water in a flask.
Mix well.
2. Cover the flask and store it at room temperature until ready for gel staining.
Wear Gloves
3. Do not stain gel(s) in the electrophoresis apparatus. and Goggles
Staining and Destaining
1. Remove the agarose gel from its bed and completely submerse the gel in a small,
clean weigh boat or lid from pipet tip rack containing 75 ml of 1x FlashBlue™ stain.
Add additional stain if needed to completely submerge the gel.
2. Stain the gel for 5 minutes.
Note: Staining the gel for longer than 5 minutes will necessitate an extended de-
staining time. Frequent changes of distilled water will expedite the process.
3. Transfer the gel to another small tray and fill it with 250 - 300 ml
of distilled water.
4. Gently agitate the tray every few minutes. Alternatively, place it

on a shaking platform.
5. Destain the gel for 20 minutes.
Dark blue bands will become visible against a light blue background. Additional
destaining may yield optimal results.
6. Carefully remove the gel from the destaining liquid and examine the gel on a Visible
Light Gel Visualization System.
METHOD 2:
Dilute FlashBlue™ 1:200 and soak gel for 4-5 hours or overnight. Gel is then ready to
visualize.
STORAGE AND DISPOSAL OF STAIN AND GEL

See Appendix H.
33
206 Experiment
Appendix
One-Step Staining and Destaining with InstaStain® Blue
Agarose gels can be stained and destained in approximately 3

F hours using InstaStain® Blue.
1. Carefully slide the agarose gel from its tray into a small,
clean tray containing at least 75 ml of distilled/deionized
One Step Wear Gloves
water or used electrophoresis buffer. The agarose gel
Stain and and Goggles
should be completely submerged.
Destain
Inst
aSta
in™
DO NOT STAIN GELS IN THE ELECTROPHORESIS APPARATUS!
Appropriate staining trays include large weigh boats and small, plastic food
containers.
2. Gently float the InstaStain® Blue card on top of the liquid with the stain (blue
side) facing toward the gel.
Each InstaStain® Blue card will stain 49 cm2 of gel (7 x 7 cm).
3. After 30 minutes, remove the InstaStain® Blue card.
4. Cover the tray with plastic wrap to prevent evaporation. Let the gel soak un-
disturbed in the liquid for at least three hours. The gel can destain overnight
if necessary.
5. Carefully remove the gel from the staining tray and document results.
Storage and Disposal of Stain and Gel

See Appendix H.
InstaStain is a registered trademark of EDVOTEK, Inc.
34
Experiment 206
Direct Staining of DNA with Instastain® Blue
Appendix
STAINING OF DNA
G
1. After electrophoresis, place the agarose gel on a flat sur-
face covered with plastic wrap. Wear Gloves
and Goggles 1
2. Wearing gloves, place the blue dye side of the InstaStain®
Blue card(s) on the gel.
3. Firmly run your fingers several times over the entire surface of the InstaStain® card Place gel on a flat
to establish good contact between the InstaStain® card and the gel. surface covered
with plastic wrap.
4. To ensure continuous contact between the gel and the InstaStain® card, place a gel
casting tray and weight, such as a small empty beaker, on top of the InstaStain®
card.
2 Inst
aSta
in™
d in g
s Pen
ent
Pat
5. Allow the InstaStain® Blue to sit on the gel for 5 to 10 minutes.

Place the InstaStain®
6. After staining, remove the InstaStain® card. If the color of the gel appears very card on the gel.
light, wet the gel surface with buffer or distilled water and place the InstaStain®
card on the gel for an additional 5 minutes.
3
DESTAINING AND VISUALIZATION OF DNA DNA InstaSt
ain™
g
Patents Pendin
1. Transfer the gel to a large weigh boat or small plastic container.

Press firmly.
2. Destain with approximately 100 ml of distilled water to cover the gel.
4 -
-
-
-
3. Repeat destaining by changing the distilled water as needed. -
Larger DNA bands will initially be visible as dark blue bands against a lighter blue InstaStain™
background. When the gel is completely destained, larger DNA bands will become
g
Patents Pendin
sharper and smaller bands will be visible. With additional destaining, the entire Place a small weight
background will become uniformly light blue. Destaining time may vary between 20 for approx. 5 minutes.
- 90 minutes.
4. Carefully remove the gel from the destain solution and examine the gel on a Visible 5
Light Gel Visualization System. To optimize visibility, use the amber filter.
5. If the gel is too light and bands are difficult to see, repeat the staining and destain-
ing procedures. Transfer to a small
tray for destaining.
Storage and Disposal of Stain and Gel 6

See Appendix H.
Destain with 37°C

distilled water.
InstaStain is a registered trademark of EDVOTEK, Inc.
35
206 Experiment
Appendix
General Notes on Staining and Destaining with FlashBlue™
Liquid stain and InstaStain® Blue Cards
H
• Use of warmed distilled water at 37° C will accelerate destaining. Destaining will
take longer with room temperature water.
• DO NOT EXCEED 37° C! Warmer temperatures will soften the gel and may cause it
to break.
• The volume of distilled water for destaining depends upon the size of the tray.
Use the smallest tray available that will accommodate the gel. The gel should be
completely submerged during destaining.
• Do not exceed 3 changes of water for destaining. Excessive destaining will cause
the bands to be very light.
Storage and Disposal of FlashBlue™ Liquid Stain, InstaStain® Blue

Cards and Gels
• Stained gels may be stored in the refrigerator for several weeks. Place the gel in a
sealable plastic bag with destaining liquid.
• DO NOT FREEZE AGAROSE GELS!
• Used InstaStain® cards and destained gels can be discarded in solid waste disposal.
• Destaining solutions can be disposed down the drain.
36
Material Safety Data Sheet Material Safety Data Sheet
May be used to comply with OSHA's Hazard Communication May be used to comply with OSHA's Hazard Communication
Material Safety Data Sheet
® Standard. 29 CFR 1910.1200 Standard must be consulted for May be used to comply with OSHA's Hazard Communication
EDVOTEK EDVOTEK ® Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements. EDVOTEK ® Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
specific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must applicable, or no information is available, the space must
Enzyme Reaction Buffer be marked to indicate that. 50x Electrophoresis Buffer be marked to indicate that. Agarose be marked to indicate that.
Section I Section I Section I
Manufacturer's Name Emergency Telephone Number Manufacturer's Name Emergency Telephone Number Manufacturer's Name Emergency Telephone Number
202-310-1500 202-370-1500 202-370-1500
EDVOTEK, Inc. EDVOTEK, Inc. EDVOTEK, Inc.
Telephone Number for information Telephone Number for information Telephone Number for information
Address (Number, Street, City, State, Zip Code) 202-310-1500 Address (Number, Street, City, State, Zip Code) Address (Number, Street, City, State, Zip Code)
202-370-1500 202-370-1500
Date Prepared Date Prepared Date Prepared
1121 5th Street NW 06/15/07 1121 5th Street NW 11-16-11 1121 5th Street NW 11-16-11
Washington DC 20001 Signature of Preparer (optional) Washington DC 20001 Washington DC 20001
Signature of Preparer (optional) Signature of Preparer (optional)
Section II - Hazardous Ingredients/Identify Information Section II - Hazardous Ingredients/Identify Information Section II - Hazardous Ingredients/Identify Information
Hazardous Components [Specific Other Limits Hazardous Components [Specific Other Limits Hazardous Components [Specific Other Limits
Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional) Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional) Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional)
This product contains no hazardous materials as defined by the OSHA Hazard This product contains no hazardous materials as defined by the OSHA Hazard This product contains no hazardous materials as defined by the OSHA Hazard Communication
Communication Standard. Communication Standard. Standard.
CAS #9012-36-6
Section III - Physical/Chemical Characteristics Section III - Physical/Chemical Characteristics Section III - Physical/Chemical Characteristics
Boiling Point Specific Gravity (H 0 = 1)
Boiling Point No data Specific Gravity (H 0 = 1) No data Boiling Point No data Specific Gravity (H 0 = 1) No data No data
2 2 For 1% solution 194 F 2
Vapor Pressure (mm Hg.) Melting Point Vapor Pressure (mm Hg.) No data Melting Point No data Vapor Pressure (mm Hg.) No data Melting Point No data
No data N/A
Evaporation Rate Evaporation Rate No data Evaporation Rate No data
Vapor Density (AIR = 1) Vapor Density (AIR = 1) No data Vapor Density (AIR = 1) No data
(Butyl Acetate = 1) No data (Butyl Acetate = 1) (Butyl Acetate = 1)
No data
Solubility in Water Solubility in Water Solubility in Water
Appreciable, (greater than 10%) Insoluble - cold
soluble
Appearance and Odor Appearance and Odor
Appearance and Odor Clear, liquid, slight vinegar odor White powder, no odor
Clear liquid, no odor, dry chemical, carbon dioxide, water spray or foam.
Section IV - Physical/Chemical Characteristics Section IV - Physical/Chemical Characteristics N.D. = No data Section IV - Physical/Chemical Characteristics N.D. = No data
Flash Point (Method Used) Flammable Limits LEL UEL LEL UEL Flash Point (Method Used) Flammable Limits LEL UEL
Flash Point (Method Used) Flammable Limits No data N.D. N.D.
No data No data No data No data N.D. N.D.
Extinguishing Media Extinguishing Media Extinguishing Media Water spray, dry chemical, carbon dioxide, halon or standard foam
Use extinquishing media appropriate to surrounding fire Use extinguishing media appropriate for surrounding fire.
Special Fire Fighting Procedures Special Fire Fighting Procedures Special Fire Fighting Procedures
Wear protective equipment and SCBA with full facepiece Possible fire hazard when exposed to heat or flame
operated in positive pressure mode.
Remove container from fire if possible.
Unusual Fire and Explosion Hazards
Unusual Fire and Explosion Hazards Unusual Fire and Explosion Hazards
None identified None
May produce toxic gases
Section V - Reactivity Data
Stability Unstable Conditions to Avoid Section V - Reactivity Data
Stability Unstable Conditions to Avoid
Section V - Reactivity Data
Stable X None Conditions to Avoid
X None Stability Unstable
Incompatibility Copper, iron, silver salts, hydrogen peroxide, phenol, picric acid
Stable
Stable X None
Formaldehyde, ether, alcohol, nitrogen oxide, strong bases, oxidizing agents. Incompatibility Strong oxidizing agents
Hazardous Decomposition or Byproducts Incompatibility No data available
Hazardous Decomposition or Byproducts
Material Safety Data Sheets
Toxic gases: Carbon monoxide, carbon dioxide, nitrogen oxides, chlorine, hydrogen chloride Carbon monoxide, Carbon dioxide
Hazardous Decomposition or Byproducts
Hazardous May Occur Conditions to Avoid
Polymerization X None Hazardous May Occur Conditions to Avoid
Will Not Occur Hazardous May Occur Conditions to Avoid
Polymerization Will Not Occur X None
Section VI - Health Hazard Data Polymerization Will Not Occur X None
Route(s) of Entry: Inhalation? Ingestion?
Section VI - Health Hazard Data
Skin? Section VI - Health Hazard Data
Yes Yes Yes Route(s) of Entry: Inhalation? Skin? Ingestion?
Yes Yes Yes Route(s) of Entry: Inhalation? Skin? Ingestion?
Health Hazards (Acute and Chronic) Toxicity has not been quantified. Sensitivity reactions (allergic) Yes Yes Yes
may occur by skin penetration including anaphylactic shock. Health Hazards (Acute and Chronic)
None Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? IARC Monographs? OSHA Regulation?
None No data No data No data NTP? IARC Monographs? Inhalation: No data available Ingestion: Large amounts may cause diarrhea
Carcinogenicity: None identified OSHA Regulation?
Carcinogenicity: NTP? IARC Monographs? OSHA Regulation?
Signs and Symptoms of Exposure
May cause irritation to skin/eye, mucous membranes and upper respiratory tract. Signs and Symptoms of Exposure
Irritation to upper respiratory tract, skin, eyes Signs and Symptoms of Exposure No data available
Medical Conditions Generally Aggravated by Exposure
Respiratory conditions Medical Conditions Generally Aggravated by Exposure None Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures No data available
Treat symptomatically and supportively Emergency First Aid Procedures Ingestion: If conscious, give large amounts of water Emergency First Aid Procedures
Eyes: Flush with water Inhalation: Move to fresh air Skin: Wash with soap and water Treat symptomatically and supportively
Section VII - Precautions for Safe Handling and Use
Steps to be Taken in case Material is Released for Spilled Section VII - Precautions for Safe Handling and Use Section VII - Precautions for Safe Handling and Use
Full-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.
Mop up with absorbant material. Dispose of properly. Steps to be Taken in case Material is Released for Spilled
Wear suitable protective clothing. Mop up spill Steps to be Taken in case Material is Released for Spilled
and rinse with water, or collect in absorptive material and dispose of the absorptive material. Sweep up and place in suitable container for disposal
Waste Disposal Method
Mix with vermiculite and dry caustic, wrap in paper and burn in a chemical incinerator equipped with Waste Disposal Method Waste Disposal Method
Dispose in accordance with all applicable federal, state, and local
afterburner and scrubber. Ignite in presence of sodium carbonate and slaked lime (CaOH) enviromental regulations. Normal solid waste disposal
Precautions to be Taken in Handling and Storing
Wear protective gear to avoid skin/eye contact Precautions to be Taken in Handling and Storing Precautions to be Taken in Handling and Storing
Avoid eye and skin contact. None
Other Precautions
None Other Precautions None Other Precautions
None
Section VIII - Control Measures
Section VIII - Control Measures Section VIII - Control Measures
Respiratory Protection (Specify Type) Chemical cartridge respirator with organic vapor cartridge.
Respiratory Protection (Specify Type) Respiratory Protection (Specify Type) Chemical cartridge respirator with full facepiece.
Ventilation Local Exhaust Special
Yes None Special
Ventilation Local Exhaust Yes None Ventilation Local Exhaust Special
Mechanical (General) No Other Process Encl. Vent. Sys.
Experiment
Mechanical (General) Yes Other None Mechanical Gen. dilution ventilation Other
206
Protective Gloves
yes Eye ProtectionSplash proof goggles
Protective Gloves Yes _Safety goggles
Eye Protection Protective Gloves Eye Protection Splash proof goggles
Yes
Other Protective Clothing or Equipment
Protection to avoid skin contact Other Protective Clothing or Equipment None Other Protective Clothing or Equipment
Impervious clothing to prevent skin contact
Work/Hygienic Practices Do not ingest. Avoid contact with skin, eyes and clothing. Wash thoroughly
after handling. Work/Hygienic Practices None Work/Hygienic Practices
None
37
38
Material Safety Data Sheet Material Safety Data Sheet
May be used to comply with OSHA's Hazard Communication May be used to comply with OSHA's Hazard Communication
EDVOTEK ® Standard. 29 CFR 1910.1200 Standard must be consulted for EDVOTEK ® Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements. specific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must applicable, or no information is available, the space must
InstaStain® Blue, FlashBlue™ be marked to indicate that. InstaStain® Ethidium Bromide be marked to indicate that.
Section I Section I
Manufacturer's Name Emergency Telephone Number Manufacturer's Name Emergency Telephone Number
202-370-1500 202-370-1500
EDVOTEK, Inc. EDVOTEK, Inc.
Telephone Number for information Telephone Number for information
Address (Number, Street, City, State, Zip Code) 202-370-1500 1121 5th Street NW 202-370-1500
Date Prepared
Washington DC 20001 Date Prepared
11-16-11
Experiment
1121 5th Street NW 11-16-11

206
Washington DC 20001 Signature of Preparer (optional) Signature of Preparer (optional)
Section II - Hazardous Ingredients/Identify Information Section II - Hazardous Ingredients/Identify Information

Hazardous Components [Specific Other Limits Hazardous Components [Specific Other Limits
Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional) Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional)
Methylene Blue Ethidium Bromide Data not available
3.7 Bis (Dimethylamino) Phenothiazin 5 IUM Chloride No data available (2,7-Diamino-10-Ethyl-9-Phenylphenanthridinium Bromide)
CAS # 61-73-4 CAS# 139-33-3
Section III - Physical/Chemical Characteristics Section III - Physical/Chemical Characteristics
Boiling Point Specific Gravity (H 0 = 1) Boiling Point No data Specific Gravity (H 0 = 1) No data
No data 2 No data 2
Vapor Pressure (mm Hg.) Vapor Pressure (mm Hg.) No data Melting Point No data
No data Melting Point No data
Evaporation Rate No data Evaporation Rate No data
Vapor Density (AIR = 1) No data No data Vapor Density (AIR = 1)
(Butyl Acetate = 1) (Butyl Acetate = 1)
Solubility in Water Solubility in Water Soluble

Soluble - cold
Appearance and Odor Chemical bound to paper, no odor Appearance and Odor Chemical bound to paper, no odor
Section IV - Physical/Chemical Characteristics Section IV - Physical/Chemical Characteristics N.D. = No data

Flash Point (Method Used) Flammable Limits LEL UEL Flash Point (Method Used) Flammable Limits LEL UEL
No data available No data No data No data N.D. N.D.
Extinguishing Media Extinguishing Media
Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam
Special Fire Fighting Procedures Special Fire Fighting Procedures

Self contained breathing apparatus and protective clothing to prevent contact Wear protective clothing and SCBA to prevent contact with skin & eyes
with skin and eyes
Unusual Fire and Explosion Hazards Unusual Fire and Explosion Hazards
Emits toxid fumes under fire conditions Emits toxic fumes
Section V - Reactivity Data Section V - Reactivity Data

Stability Unstable Conditions to Avoid Stability Unstable Conditions to Avoid
Stable X None Stable X None

Incompatibility Incompatibility Strong oxidizing agents
Strong oxidizing agents
Hazardous Decomposition or Byproducts Toxic fumes of Carbon monoxide, Carbon dioxide, Hazardous Decomposition or Byproducts
nitrogen oxides, sulfur oxides, hydrogen, chloride gas Carbon monoxide, Carbon dioxide, nitrogen oxides, hydrogen bromide gas
Hazardous May Occur Conditions to Avoid Hazardous May Occur Conditions to Avoid
Polymerization Polymerization Will Not Occur X None
Will Not Occur X None
Section VI - Health Hazard Data Section VI - Health Hazard Data
Route(s) of Entry: Inhalation? Skin? Ingestion? Route(s) of Entry: Inhalation? Yes Skin?
Yes Yes Yes Yes Ingestion? Yes
Health Hazards (Acute and Chronic) Health Hazards (Acute and Chronic) Chronic: May alter genetic material
Skin: May cause skin irritation Eyes: May cause eye irritation Inhalation: Cyanosis Acute: Material irritating to mucous membranes, upper respiratory tract, eyes, skin
Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Carcinogenicity: No data available NTP? IARC Monographs? OSHA Regulation?
Meets criteria for proposed OSHA medical records rule PEREAC 47.30420.82
Signs and Symptoms of Exposure Signs and Symptoms of Exposure
No data available Irritation to mucous membranes and upper respiratory tract
Material Safety Data Sheets
Medical Conditions Generally Aggravated by Exposure No data available Medical Conditions Generally Aggravated by Exposure No data
Emergency First Aid Procedures Emergency First Aid Procedures

Treat symptomatically Treat symptomatically and supportively
Section VII - Precautions for Safe Handling and Use Section VII - Precautions for Safe Handling and Use
Steps to be Taken in case Material is Released for Spilled Steps to be Taken in case Material is Released for Spilled
Ventilate area and wash spill site Wear SCBA, rubber boots, rubber gloves
Waste Disposal Method Mix material with a combustible solvent and burn in chemical Waste Disposal Method
Mix material with combustible solvent and burn in a chemical incinerator
incinerator equipped with afterburner and scrubber. Check local and state regulations. equipped afterburner and scrubber
Precautions to be Taken in Handling and Storing Precautions to be Taken in Handling and Storing
Keep tightly closed. Store in cool, dry place Use in chemical fume hood with proper protective lab gear.
Other Precautions Other Precautions

None Mutagen
Full-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.

Respiratory Protection (Specify Type) Respiratory Protection (Specify Type) SCBA
MIOSH/OSHA approved, SCBA
Special Ventilation Local Exhaust Yes Special Chem. fume hood
Ventilation Local Exhaust
Mechanical (General) Required Other Mechanical (General) No Other None
Protective Gloves Eye Protection Protective Gloves Rubber Eye Protection Chem. safety goggles
Rubber Chem. safety goggles
Other Protective Clothing or Equipment Rubber boots Other Protective Clothing or Equipment Rubber boots
Work/Hygienic Practices Work/Hygienic Practices

Use in chemical fume hood with proper protective lab gear.

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The Biotechnology Education Company ®

Storage: See Page 3 for

EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com

Material Safety Data Sheets 37

EDVOTEK, The Biotechnology Education Company, and InstaStain are registered

THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA.

The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Reagents & Supplies

Non Frost-free Freezer

EDVOTEK - The Biotechnology Education Company®

• Horizontal gel electrophoresis apparatus

• Experiment number and title

Visit our web site for information

The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

A+B A+C B+C A+B+C 500

Generally, a restriction enzyme map is constructed by first determining the number

This can be visually demonstrated by a description of an example as shown in Figure

log of their molecular weight.

Standard DNA fragments, which were

Dimer Trimer 23130 9416 6557

Experiment Overview and General Instructions

LABORATORY SAFETY GUIDELINES:

2. Exercise caution when working in the laboratory –

3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.

5. If you are unsure of something, ASK YOUR INSTRUCTOR!

Before starting the Experiment:

During the Experiment:

After the Experiment:

3. Use an automatic micropipet to dispense 30 µl of Rxn Buffer (enzyme reaction buf-

7. Incubate the tubes in a 45° C waterbath for 15 minutes or in a 37° C waterbath

Chart II: Options for

Restriction Enzyme Reactions

After incubation is completed: Important

The prepared DNA samples can be stored in the

freezer for electrophoresis at a later time.

Prepare the Gel (see Appendices A, B, and C):

• Agarose gel concentration required: 0.8% Wear

• Well-former template (comb)

Agarose Gel Electrophoresis

Stain the Gel:

DO NOT STAIN GELS IN THE ELECTROPHORESIS APPARATUS!

View the Gel:

2. Label the semi-log graph paper:

• Label the non-logarithmic horizontal x-axis "Migration Distance" in centimeters

• Label the logarithmic vertical y-axis "Log base

size in base pairs, on the y-axis. 10,000 base pairs

points scattered on each side of the line. Some 4

points may be right on the line (see example at left). 3

5. Measure migration distance of each of the DNA 2

fragments from the three restriction enzyme

from the restriction enzyme digestion reactions 5

• Find migration distance of unknown frag- 3

ment on x-axis - draw vertical line from that

• From the point of intersection, draw a second 100

Size Determination of DNA Restriction Fragments

MAPPING RESTRICTION SITES ON PLASMID DNA

The size of the plasmid used in this experiment is 4340 bp.

1. Draw a circle representing a 4340 bp plasmid on a transparent sheet of acetate.

2. Draw a second circle representing a 4340 bp plasmid on a transparent sheet of

• Specify, in base pairs, the distances between all the sites.

4. Compare fragment sizes of the single digests of HindIII and Bgl I

X-axis: Migration distance (cm)

Answer the following study questions in your laboratory notebook or on a separate