DNA Fingerprinting

The Biotechnology Education Company
EDVOTEK, Inc. 1-800-EDVOTEK www.edvotek.com

EVT 100202AM
EDVO-Kit
109
DNA Fingerprinting:
Identication of
DNA Restriction
Fragmentation Patterns
See Page 3 for storage instructions.
EXPERIMENT OBJECTIVE:
The objective of this experiment is to develop a basic understanding of
DNA ngerprinting. Variations in restriction enzyme cleavage patterns
obtained from different DNA molecules will be analyzed and the
possible perpetrator of a crime will be identied using
the logic of DNA ngerprinting.
2
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns
EVT 100202AM
All components are intended for educational
research only. They are not to be used for diag-
nostic 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.
EDVOTEK, The Biotechnology Education Company, and InstaStain are registered
trademarks of EDVOTEK, Inc.. Ready-to-Load, UltraSpec-Agarose and FlashBlue
are trademarks of EDVOTEK, Inc.
Page
Experiment Components 3
Experiment Requirements 3
Background Information 4
Experiment Procedures
Experiment Overview and General Instructions 10
Agarose Gel Electrophoresis 12
Study Questions 13

Instructor's Guidelines
Notes to the Instructor and Pre-Lab Preparations 15
Experiment Results and Analysis 21
Study Questions and Answers 22
Appendices 23
Material Safety Data Sheets 34
Table of Contents
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Experiment
DNA Fingerprinting - ID of DNA
Restriction Fragmentation Patterns
EVT 100202AM
EDVOTEK - The Biotechnology Education Company
1-800-EDVOTEK www.edvotek.com
FAX: (301) 340-0582 email: info@edvotek.com
READY-TO-LOAD DNA SAMPLES
FOR ELECTROPHORESIS
A DNA from crime scene cut with Enzyme 1
B DNA from crime scene cut with Enzyme 2
C DNA from Suspect 1 cut with Enzyme 1
D DNA from Suspect 1 cut with Enzyme 2
E DNA from Suspect 2 cut with Enzyme 1
F DNA from Suspect 2 cut with Enzyme 2
REAGENTS & SUPPLIES
UltraSpec-Agarose powder
Concentrated electrophoresis buffer
FlashBlue DNA Stain
InstaStain Blue cards
Practice Gel Loading Solution
1 ml pipet
Microtipped Transfer Pipets
Note: If you ordered Experiment #109-Q, the experiment components in-
clude InstaStain Ethidium bromide instead of FlashBlue and InstaStain
Blue DNA stains.
DNA samples are stable at
room temperature. However,
if the experiment will not be
conducted within one month of
receipt, it is recommended that
the DNA samples be stored in
the refrigerator.
DNA samples do not require
heating prior to gel loading.
Experiment Components
Requirements
Horizontal gel electrophoresis apparatus
D.C. power supply
Automatic micropipets with tips
Balance
Microwave, hot plate or burner
Pipet pump
250 ml asks or beakers
Hot gloves
Safety goggles and disposable laboratory gloves
Small plastic trays or large weigh boats (for gel destaining)
DNA visualization system (white light)
Distilled or deionized water
4
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 1989,1992,1994,1997,1998, 2000, 2004, 2007, 2009, EDVOTEK, Inc., all rights reserved. EVT 100202AM
109
Experiment
Background Information
DNA typing (also called DNA prole analysis or DNA ngerprinting) is the process where-
by the genomic DNA of an organism is analyzed by examining several specic, variable
DNA sequences located throughout the genome. In humans, DNA ngerprinting is now
used routinely for identication purposes.
Human DNA ngerprinting was pioneered by Dr. Alex Jeffreys at the University of Leices-
ter in 1984 which led to the apprehension of a murderer in the rst DNA ngerprinting
conviction in September 1987 in the UK. Two months later, the rst U.S. conviction based
on DNA ngerprinting occurred in Orlando, Florida. Since then, the use of DNA nger-
printing has led to thousands of criminal convictions, as well as dozens of exonerations.
In contrast to earlier methodologies, such as blood typing which can only exclude a sus-
pect, DNA ngerprinting can provide positive identication with great accuracy. In addi-
tion to criminal identication cases, DNA ngerprinting is now used routinely in paternity
determinations and for the identication of genetic disease "markers". It is also used for
the identication of human remains, such as in war casualties, and was used extensively
to identify victims of the September 11, 2001 terrorist attacks on the World Trade Cen-
ter, the Pentagon, and passengers in the plane which crashed in a eld near Shanksville,
Pennsylvania.
Human cells contain two types of DNA. The rst type is cellular chromosomal DNA, which
is packaged in 23 sets of chromosomes in the nucleus of the cell. This DNA, obtained
from both parents, reects the combined parental genetic inheritance of an individual.
DNA ngerprinting utilizing cellular DNA involves analysis of the sequence of two alleles
for a particular gene.
The second type of DNA is different from cellular DNA and is present only in the mito-
chondria, which are the energy-producing organelles of the cell. Mitochondrial DNA is
inherited maternally by both males and females and is extremely useful in the analysis of
specic cases where fraternal linkages are important to determine. For example, a broth-
er, sister, half brother or half sister who share the same mother would inherit the same
mitochondrial DNA. Identication is determined by sequencing certain regions within
mitochondrial DNA, which is a single circular chromosome composed of 16,569 base pairs.
DNA ngerprinting developed by Dr. Jeffreys utilizes cellular chromosomal DNA submit-
ted to restriction enzyme digestion and Southern blot analysis. When human DNA is
digested by a restriction enzyme, a very large number of DNA fragments are generated.
When separated by agarose gel electrophoresis, the numerous DNA fragments appear
as a "smear" on the gel. Labeled probes are used to detect Restriction Fragment Length
Polymorphic (RFLP) regions within DNA, which will be described in greater detail. DNA
RFLP analysis is statistically very accurate but requires relatively large amounts of DNA
and takes several days to perform.
In recent years, the use of the RFLP method has been overtaken by the Polymerase Chain
Reaction (PCR) method because of two important advantages. The rst is the sensitivity
of PCR, which allows for DNA ngerprinting identication using much smaller amounts
of DNA. This is because PCR is able to amplify DNA to facilitate analysis. The second ad-
vantage is the speed of PCR analysis, which allows critical questions to be answered more
quickly compared to Southern Blot analysis. One PCR cycle has three steps, resulting in a
doubling of the amount of DNA (see Figure 1).
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The Polymerase Chain Reaction (PCR) method amplies target sequences of DNA, which
are referred to as AMRFLPs. PCR made it possible for very small amounts of DNA found
at crime scenes to be amplied for DNA ngerprinting analysis. A specic set of two
primers is used to prime DNA polymerase to synthesize many copies of the targeted areas
of DNA.
Many important concepts of molecular biology can be conveyed in the context of DNA
Fingerprinting methods. In this experiment, emphasis is placed on concepts related to
RFLP analysis. The experiment activities will focus on the identication of DNA by ana-
lyzing restriction fragmentation patterns separated by agarose gel electrophoresis.
3' 5'
5' 3'
5' 3'
3' 5'
5' 3'
5'
5'

Denature 94C
Extension
72C
3' 5'
Separation of
2 DNA strands
=
Primer 1 =
Primer 2 =

5' 3'
5'
Anneal
2 primers
45C
3' 5'
5'
C
y
c
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1

Target Sequence
Figure 1: The Polymerase Chain Reaction
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Experiment
USE OF RESTRICTION ENZYMES IN DNA FINGERPRINTING
DNA ngerprinting involves the electrophoretic analysis of DNA fragment sizes gener-
ated by restriction enzymes. Restriction enzymes are endonucleases which catalyze the
cleavage of phosphodiester bonds within both DNA strands. The sites of cleavage occur
in or near very specic palindromic sequences of bases called recognition sites, which are
generally 4 to 8 base pairs in length.
The two most commonly used restriction enzymes for DNA prole analysis are Hae III and
Hinf I, which are 4-base and 5-base cutting enzymes. The examples in the gure 2 show
recognition sites for various restriction enzymes.
The size of the DNA fragments generated depends on
the distance between the recognition sites. In general,
the longer the DNA molecule, the greater the probabil-
ity that a given recognition site will occur. Human DNA
is very large and contains approximately three billion
base pairs. A restriction enzyme having a 6-base pair
recognition site, such as Eco RI, would be expected to
cut human DNA into approximately 750,000 different
fragments.
DNA is highly polymorphic - that is, no two individuals
have exactly the same pattern of restriction enzyme
recognition sites in their DNAs. A large number of al-
leles exist in the population. Alleles, which are alter-
nate forms of a gene, result in alternative expressions of
genetic traits which can be dominant or recessive.
Chromosomes occur in matching pairs, one of maternal
and the other of paternal origin. The two copies of a gene (alleles) at a given chromo-
somal locus represent a composite of the parental genes constituting an individuals
unique genotype. It follows that alleles have differences in their base sequences which
consequently creates differences in the distribution and frequencies of restriction en-
zyme recognition sites. Other differences in base sequences between individuals can
occur because of mutations and deletions. Such changes can also create or eliminate a
recognition site.
Polymorphic DNA refers to chromosomal regions that vary widely from individual to in-
dividual. By examining several of these regions within the genomic DNA obtained from
an individual, one may obtain a "DNA ngerprint" for that individual. The most com-
monly used polymorphisms are those that vary in length; these are known as Fragment
Length Polymorphisms (FLPs). The main reason for the occurrence of RFLPs is because of
variations in length of a given segment of genomic DNA between two restriction enzyme
recognition sites among individuals of the same species.
5'....GGCC....3'
3'....CCGG....5'
Hae III
5'....GGATCC....3'
3'....CCTAGG....5'
Bam HI
5'....CTGCAG....3'
3'....GACGTC....5'
Pst I
5'....GANTC....3'
3'....CTNAG....5'
Hinf I
Figure 2: Restriction enzyme recognition sites
7
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Likewise, RFLP can occur in "intergenic" or noncoding regions of DNA and is known as
Variable Number of Tandem Repeats (VNTRs). In this case, segments of DNA that contain
sequences from 2 to 40 bases in length repeat in tandem manner many times. The num-
ber of segments or "core unit" repeats varies among individuals of the same species while
the restriction enzyme cut sites are not altered. VNTR loci are very polymorphic. There
are potentially hundreds of alleles at a single locus and therefore they are very useful in
DNA ngerprinting. Ten to fteen percent of mammalian DNA consists of sets of repeat-
ed, short sequences of bases that are tandemly arranged in arrays. The length of these
arrays (the amount of repeated sets) varies between individuals at different chromosomal
loci.
TGTTTA
|
TGTTTA
|
TGTTTA.........variable number
When these sequences in DNA are anked by recognition sites, the length of the repeat
will determine the size of the restriction enzyme fragment generated. There are several
types of these short, repetitive sequences and they have been characterized.
DNA FINGERPRINTING USING SOUTHERN BLOTS
Agarose gel electrophoresis is a procedure used to analyze DNA fragments generated by
restriction enzymes. The gel consists of microscopic pores that act as a molecular sieve.
Samples of DNA are loaded into wells made in the gel during casting. Since DNA has a
negative charge at neutral pH, it migrates through the gel towards the positive electrode
during electrophoresis. DNA fragments are separated by the gel according to their size.
The smaller the fragment the faster it migrates. After electrophoresis, the DNA can be
visualized by staining the gel with dyes. Restriction enzyme cleavage of relatively small
DNA molecules, such as plasmids and viral DNAs, usually results in discrete banding pat-
terns of the DNA fragments after electrophoresis. However, cleavage of large and com-
plex DNA, such as human chromosomal DNA, generates so many differently sized frag-
ments that the resolving capacity of the gel is exceeded. Consequently, the cleaved DNA
is visualized as a smear after staining and has no obvious banding patterns.
RFLP analysis of genomic DNA is facilitated by Southern Blot analysis. After electro-
phoresis, the DNA fragments in the gel are denatured by soaking in an alkali solution.
This causes double-stranded DNA fragments to be converted into single-stranded form
(no longer base-paired in a double helix). A replica of the electrophoretic pattern of
DNA fragments in the gel is made by transferring (blotting) them to a sheet of nylon
membrane. This is done by placing the membrane on the gel after electrophoresis and
transferring the fragments to the membrane by capillary action or suction by vacuum.
The DNA, which is not visible, becomes permanently adsorbed to the membrane, and can
be manipulated easier than gels.
8
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Experiment
Analysis of the blotted DNA is done by hybrid-
ization with a labeled DNA probe. In forensic
RFLP analysis, the probe is a DNA fragment that
contains base sequences which are complemen-
tary to the variable arrays of tandemly repeated
sequences found in the human chromosomes.
Probes can be labeled with isotopic or non-iso-
topic reporter molecules, such as uorescent
dyes used for detection. A solution containing
the single-stranded probe is incubated with
the membrane containing the blotted, single-
stranded (denatured) DNA fragments. Under
the proper conditions, the probe will only base
pair (hybridize) to those fragments containing
the complementary repeated sequences. The
membrane is then washed to remove excess
probe. If the probe is isotopically labeled to the
membrane, it is then placed on an x-ray lm for
several hours. This process is known as autoradi-
ography. Only DNA fragments that have hybrid-
ized to the probe will reveal their positions on
the lm because the localized areas of radioac-
tivity cause exposure. The hybridized fragments
appear as discrete bands (ngerprint) on the lm
and are in the same relative positions as they
were in the agarose gel after electrophoresis.
Only specic DNA fragments, of the hundreds of
thousands of fragments present, will hybridize
with the probe because of the selective nature of
the hybridization (base pairing) process.
In forensic cases, DNA samples can be extracted
and puried from small specimens of skin, blood,
semen, or hair roots collected at the crime scene.
DNA that is suitable for analysis can also be
obtained from dried stains of semen and blood.
The RFLP analyses performed on these samples
is then compared to samples obtained from the
suspect. If the RFLP patterns match, it is then
beyond reasonable doubt that the suspect was
at the crime scene. In practice, several different
probes containing different types of repetitious
sequences are used in the hybridizations in order
to satisfy certain statistical criteria for absolute,
positive identication. To assure positive iden-
tication in criminal cases, 13 different loci are
compared between a suspect and evidence DNA
obtained from the crime scene.
Probe overlaps both the variable region, as
well as adjacent part of the genome. Arrows
show restriction enzyme sites with probe for
Southern Blot analysis. PCR can also be used to
detect variable nucleotide regions.
Lane 1 DNA Marker
Lane 2 Homozygous Copies
Lane 3 Heterozygous VNTR
(Lanes 3, 4, and 6 represent different combina-
tions of the three VNTRs.)
Probe
Probe
Probe
A
B
C
1 2 3 4 5 6 7
30 Kb
40 Kb
50 Kb
50 Kb
40 Kb
30 Kb A
B
C
Genotypes
Figure 3: RFLP analysis demonstrating
Variable Numbers of Nucleotide Tandem
Repeats (VNTR).
9
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In this experiment, DNAs are pre-digested by restriction enzymes and the fragmentation
patterns serve as the individual ngerprint. The DNA fragmentation patterns can be
analyzed directly in the stained agarose gel, which eliminates the need for a Southern
blot. In this hypothetical case, DNA obtained from two suspects are cleaved with two
restriction enzymes in separate reactions. The objective is to analyze and match the DNA
fragmentation patterns after agarose gel electrophoresis and determine if Suspect 1 or
Suspect 2 was at the crime scene.
THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA.

4
5
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Autoradiograph
2
3
1
Evidence
Suspect's
Blood
1. Collection of DNA
2. Extraction of DNA
3. DNA cut into fragments by restriction
enzymes
4. DNA fragments separated by agarose
gel electrophoresis
5. DNA denatured into single strands
6. Blot DNA onto a nylon membrane
(Southern Blot)
7. Nylon membrane soaked with
probes that bind to target DNA
fragments and detected.
8. Computer analysis
6
Southern Blot
Figure 4 DNA Fingerprinting
by RFLP Analysis
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Experiment Overview and General Instructions
EXPERIMENT OBJECTIVE:
The objective of this experiment is to develop a basic understanding of DNA ngerprint-
ing. Variations in restriction enzyme cleavage patterns obtained from different DNA
molecules will be analyzed and the possible perpetrator of a crime will be identied using
the logic of DNA ngerprinting.
LABORATORY SAFETY
1. Gloves and goggles should be worn routinely as good
laboratory practice.
2. Exercise extreme caution when working with equipment that is used in conjunction
with the heating and/or melting of reagents.
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4. Exercise caution when using any electrical equipment in
the laboratory.
5. Always wash hands thoroughly with soap and water
after handling reagents or biological materials in the
laboratory.
LABORATORY NOTEBOOK RECORDINGS:
Address and record the following in your laboratory notebook or on
a separate worksheet.
Before starting the Experiment:
Write a hypothesis that reects the experiment.
Predict experimental outcomes.
During the Experiment:

Record (draw) your observations, or photograph the results.
Following the Experiment:

Formulate an explanation from the results.
Determine what could be changed in the experiment if the experiment
were repeated.
Write a hypothesis that would reect this change.
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After electrophoresis,
transfer gel for staining
Analysis
on white
light
source
FlashBlue
DNA stain
Attach safety
cover,connect
leads to power
source and conduct
electrophoresis
Load each
sample in
consecutive wells
Remove end
blocks & comb,
then submerge
gel under buffer in
electrophoresis
chamber
Prepare
agarose gel in
casting tray
6
5
4
3
2
1
Gel pattern will vary
depending upon experiment.
Experiment Overview: Flow Chart
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Prepare the Gel
1. Prepare an agarose gel with specications
summarized below. Your instructor will
specify which DNA stain you will be using.
Agarose gel concentration required: 0.8%
Recommended gel size: 7 x 7 cm or 7 x 14 cm (two gels)
Number of sample wells required: 6
Placement of well-former template: rst set of notches ( 7 x 7 cm)
rst & third set of notches
(7 x 14 cm)
Reminders:
During electrophoresis,
the DNA samples migrate
through the agarose gel to-
wards the positive electrode.
Before loading the samples,
make sure the gel is properly
oriented in the apparatus
chamber.
+
Black Red
Sample wells
Agarose Gel Electrophoresis

Lane Tube
A DNA from crime scene cut with Enzyme 1
B DNA from crime scene cut with Enzyme 2
C DNA from Suspect 1 cut with Enzyme 1
D DNA from Suspect 1 cut with Enzyme 2
E DNA from Suspect 2 cut with Enzyme 1
F DNA from Suspect 2 cut with Enzyme 2
Run the Gel
3. After DNA samples are loaded, connect the apparatus to the D.C. power
source and set the power source at the required voltage.
4. Check that current is owing properly - you should see bubbles forming
on the two platinum electrodes. Conduct electrophoresis for the length of
time specied by your instructor.
5. After electrophoresis is completed, proceed to DNA staining and visualiza-
tion. Refer to Appendix E, F, G, or H for the appropriate staining instruc-
tions.
6. Document the results of the gel by photodocumentation.
Alternatively, place transparency lm on the gel and trace it with a permanent
marking pen. Remember to include the outline of the gel and the sample
wells in addition to the migration pattern of the DNA bands.
For gels to be stained with
FlashBlue or InstaStain
Blue, prepare gels accord-
ing to Appendix A.
For gels to be stained
with InstaStain Ethidium
bromide, prepare gels ac-
cording to Appendix B.
Step-by-step guidelines
for agarose gel prepara-
tion are summarized in
Appendix D.
Wear
Gloves & goggles
Load the Samples
2. Load the DNA samples in tubes A - F into the wells in consecutive order.
For gels to be stained with FlashBlue or InstaStain Blue, ll wells
with 35 - 38 l.
For gels to be stained with InstaStain Ethidium Bromide, ll wells
with 18 - 20 l.
13
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1. Dene FLPs and give their signicance.
2. What is the most likely cause of Restriction Fragment Length Polymorphisms?
3. What are Variable Number of Tandem Repeats (VNTRs)?
4. Who are the only individuals possessing the same DNA ngerprints?
5. List the steps involved in DNA ngerprinting from extraction of DNA through the
matching of a suspect to a crime scene sample.
6. What type of human cells can be utilized for this technique?
Study Questions
14
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Experiment
EVT 100202AM
Instructors
Guide
Class size, length of laboratory sessions, and availability of equipment are
factors which must be considered in planning and implementing this experi-
ment with your students. These guidelines can be adapted to t your spe-
cic set of circumstances. If you do not nd the answers to your questions
in this section, a variety of resources are continuously being added to the
EDVOTEK web site. 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).
EDUCATIONAL RESOURCES, NATIONAL CONTENT AND SKILL
STANDARDS
By performing this experiment, students will learn to load samples and run
agarose gel electrophoresis. Experiment analysis will provide students the
means to transform an abstract concept into a concrete explanation.
Notes to the Instructor & Pre-Lab Preparations
Visit our web site for information
about EDVOTEK's complete line
of experiments for biotechnology
and biology education.
EDVOTEK Ready-to-Load Electrophoresis Ex-
periments are easy to perform and are designed
for maximum success in the classroom setting.
However, even the most experienced students
and teachers occasionally encounter experimen-
tal problems or difculties. EDVOTEK web site
resources provide suggestions and valuable hints
for conducting electrophoresis, as well as answers
to frequently asked electrophoresis questions.
Laboratory Extensions and Supplemental
Activities
Laboratory extensions are easy to perform using
EDVOTEK experiment kits. For example, a DNA
sizing determination activity can be performed
on any electrophoresis gel result if DNA markers
are run in parallel with other DNA samples. For
DNA Sizing instructions, and other laboratory ex-
tension suggestions, please refer to the EDVOTEK
website.
Visit the EDVOTEK web site often for
continuously updated information.
M
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- Fri 9 a
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p
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(1-800-338-6835)
E
D
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CH SERVIC
E
1-800-EDVOTEK
Mon - Fri
9:00 am to 6:00 pm ET
FAX: (301) 340-0582
Web: www.edvotek.com
email: edvotek@aol.com
Please have the following
information ready:
Experiment number and title
Kit lot number on box or tube
Literature version number
(in lower right corner)
Approximate purchase date
Technical Service
Department
Order
Online
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Instructors Guide
APPROXIMATE TIME REQUIREMENTS
1. Gel preparation:
Whether you choose to prepare the gel(s) in advance or have the students prepare
their own, allow approximately 30 minutes for this procedure. Generally, 20 minutes
of this time is required for gel solidication.
2. Micropipeting and Gel Loading:
If your students are unfamiliar with using micropipets and sample loading tech-
niques, a micropipeting or practice gel loading activity is suggested prior to conduct-
ing the experiment. Two suggested activities are:
EDVOTEK Expt. # S-44, Micropipetting Basics, focuses exclusively on using micro-
pipets. Students learn pipeting techniques by preparing and delivering various
dye mixtures to a special Pipet Card.
Practice Gel Loading: EDVOTEK Series 100 electrophoresis experiments contain a
tube of practice gel loading solution for this purpose. It is highly recommended
that a separate agarose gel be cast for practice sample delivery. This activity can
require anywhere from 10 minutes to an entire laboratory session, depending
upon the skill level of your students.
3. Conducting Electrophoresis:
The approximate time for electrophoresis will vary from
approximately 15 minutes to 2 hours. Different models of
electrophoresis units will separate DNA at different rates
depending upon its design conguration. Generally, the
higher the voltage applied the faster the samples migrate.
However, maximum voltage should not exceed the indicated
recommendations. The Table C example at left shows Time
and Voltage recommendations. Refer to Table C in Appendi-
ces A or B for specic experiment guidelines.
PREPARING AGAROSE GELS FOR ELECTROPHORESIS
There are several options for preparing agarose gels for the electrophoresis experiments:
1. Individual Gel Casting: Each student lab group can be responsible for casting their
own individual gel prior to conducting the experiment.
2. Batch Gel Preparation: A batch of agarose gel can be prepared for sharing by the
class. To save time, a larger quantity of UltraSpec-Agarose can be prepared for shar-
ing by the class. See instructions for "Batch Gel Preparation".
3. Preparing Gels in Advance: Gels may be prepared ahead and stored for later use.
Solidied gels can be stored under buffer in the refrigerator for up to 2 weeks.
Do not store gels at -20C. Freezing will destroy the gels.
Time and Voltage
Recommendations
Minimum / Maximum
Volts
150
125
70
50
15 / 20 min
20 / 30 min
35 / 45 min
50 / 80 min
Table
C
EDVOTEK Electrophoresis Model
M6+ M12 & M36
Minimum / Maximum
25 / 35 min
35 / 45 min
60 / 90 min
95 / 130 min
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* 0.77 UltraSpec-Agarose gel percentage rounded up to 0.8%
Amt of
Agarose
(g)
Concentrated
Buffer (50x)
(ml)
Size of Gel
(cm)
Distilled
Water
(ml)
Total
Volume
(ml)
7 x 7
7 x 10
7 x 14
0.23
0.39
0.46
0.6
1.0
1.2
29.4
49.0
58.8
+
= +
Individual 0.8%* UltraSpec-Agarose Gel
DNA Staining with FlashBlue or InstaStain Blue
Table
A.1
30
50
60
Table
A.2
Amt of
Agarose
(g)
Diluted
Buffer (1x)
(ml)
Size of Gel
(cm)
7 x 7
7 x 10
7 x 14
0.23
0.39
0.46
30
50
60
+
Individual 0.8%*
UltraSpec-Agarose Gel
DNA Staining with FlashBlue
or InstaStain Blue
If preparing a 0.8% gel with
concentrated (50x) buffer, use Table A.1
diluted (1x) buffer, use Table A.2
USING AGAROSE GELS THAT HAVE BEEN PREPARED IN ADVANCE
If gels have been removed from their trays for storage, they should be "anchored" back
to the tray with a few drops of hot, molten agarose before placing the gels into the
apparatus for electrophoresis. This will prevent the gel from sliding around in the tray
and/or oating around in the electrophoresis chamber.
AGAROSE GEL CONCENTRATION AND VOLUME
Gel concentration is one of many factors which affect the mobility of molecules during
electrophoresis. Higher percentage gels are sturdier and easier to handle. However, the
mobility of molecules and staining will take longer because of the tighter matrix of the
gel. Gel volume varies depending on the size of the casting tray, as well as the type of
stain to be used for DNA staining after electrophoresis. Gels which will be stained with
InstaStain Ethidium Bromide require less sample amount (volume) than gels that will be
stained with FlashBlue or InstaStain Blue.
This experiment requires a 0.8% gel. It is a common agarose gel concentration for sepa-
rating dyes or DNA fragments in EDVOTEK experiments.
Specications for preparing a 0.8% gel to be stained with FlashBlue or In-
staStain Blue can be found in Appendix A.
Specications for preparing a 0.8% gel to be stained with InstaStain Ethidium
bromide can be found in Appendix B.
Tables A-1 and A-2 below are examples of tables from Appendix A. The rst (left) table
shows reagent volumes using concentrated (50x) buffer. The second (right) table shows
reagent volumes using diluted (1x) buffer.
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GEL STAINING AND DESTAINING AFTER ELECTROPHORESIS

DNA stains FlashBlue and InstaStain Blue are included in EDVOTEK standard
Series 100 experiments. For Series 100-Q experiments, InstaStain Ethidium Bromide
(InstaStain EtBr) is included. InstaStain is a proprietary staining method which
saves time and reduces liquid waste. EDVOTEK also offers Protein InstaStain for
staining Protein polyacrylamide gels, which can be purchased separately.
Instructions for DNA staining options are provided in the Appendices section.
Option 1: FlashBlue liquid - Appendix E.
This simple and rapid liquid staining and destaining procedure yields excellent visibil-
ity of DNA bands in less than 25 minutes (5 minutes staining, 20 minutes destaining).

Option 2: InstaStain Blue cards, One-step Staining and Destaining- Appendix F.
Agarose gels can be stained and destained in one easy step.

Option 3: InstaStain Blue cards - Appendix G.
Using InstaStain Blue cards, staining is completed in approximately 5-10 minutes.
DNA bands will become visible after destaining for approximately 20 minutes.
Results 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 contrasting against a uniformly light blue background.
Option 4: InstaStain Ethidium Bromide - Appendix H
Staining with ethidium bromide is very sensitive and can detect as little as 5 to 10
nanograms of DNA with the use of a U.V. transilluminator. Ethidium Bromide is a
dye that is commonly used by scientic researchers. It is a listed mutagen and forms a
tight complex with DNA by intercalating between the bases within the double helix.
The complex strongly uoresces when exposed to ultraviolet light.
CAUTION: Ethidium Bromide is a listed mutagen. Disposal of the InstaStain EtBr
cards, which contain microgram amounts of ethidium bromide, is minimal compared
to the large volume of liquid waste generated by traditional ethidium bromide stain-
ing procedures. Disposal of InstaStain cards and gels should follow institutional
guidelines for chemical waste.
19
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READY-TO-LOAD DNA SAMPLES FOR ELECTROPHORESIS
No heating required before gel loading.

EDVOTEK offers the widest selection of electrophoresis experiments which minimize
expensive equipment requirements and save valuable time for integrating important
biotechnology concepts in the teaching laboratory. Series 100 experiments feature
DNA samples which are predigested with restriction enzymes and are stable at room
temperature. DNA samples are ready for immediate delivery onto agarose gels for
electrophoretic separation and do not require pre-heating in a waterbath.
Electrophoresis samples and reagents in EDVOTEK experiments are packaged in various
formats. The samples in Series 100 and S-series electrophoresis experiments will be
packaged in one of the following ways:
1) Pre-aliquoted Quickstrip connected tubes
OR
2) Individual 1.5 ml (or 0.5 ml) microtest tubes
SAMPLES FORMAT: PRE-ALIQUOTED QUICKSTRIP
CONNECTED TUBES
Convenient QuickStrip connected tubes contain pre-aliquoted
ready-to-load samples. The samples are packaged in a microtiter
block of tubes covered with a protective overlay. Separate the
microtiter block of tubes into strips for a complete set of samples
for one gel.
1. Use sharp scissors to separate the block of samples
into individual strips as shown in the diagram at
right.
Each row of samples (strip) constitutes a complete
set of samples for each gel. The number of samples
per set will vary depending on the experiment.
Some tubes may be empty.
2. Cut carefully between the rows of samples. Do
not cut or puncture the protective overlay directly
covering the sample tubes.
F E D C B A
Carefully cut between
each set of tubes
E
D
V
O
T
E
K

D
O

N
O
T

B
E
N
D
A
B
C
D
E
F
G
H
C
U
T

H
E
R
E
A
B
C
D
E
F
G
H
C
U
T

H
E
R
E
A
B
C
D
E
F
G
H
C
U
T

H
E
R
E
C
U
T

H
E
R
E
A
B
C
D
E
F
G
H
C
U
T

H
E
R
E
A
B
C
D
E
F
G
H
A
B
C
D
E
F
G
H
3. Each gel will require one strip of samples.
4. Remind students to tap the tubes before gel loading to ensure that all of the
sample is at the bottom of the tube.
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SAMPLES FORMAT: INDIVIDUAL 1.5 ML MICROTEST TUBES
It is recommended that samples packaged in 1.5 ml individual microtest tubes be aliquot-
ed for each gel. DNA Samples packaged in this format are available in three standard
quantities:
Standard experiment kit 240 l
Bulk B-Series 480 l
Bulk C Series 960 l
1. Check all sample volumes for possible evaporation. Samples will become more con-
centrated if evaporation has occurred.
2. If needed, tap or centrifuge the sample tubes. Then add distilled water to slightly
above the following level:
1.3 cm level for Standard experiment kit
1.9 cm level for the B-Series
2.8 cm level for the C-Series
3. Mix well by inverting and tapping the
tubes several times.
4. After determining that the samples are
at their proper total volumes, aliquot
each sample into appropriately labeled
0.5 ml or 1.5 ml microtest tubes.
For gels to be stained with Flash-
Blue or InstaStain Blue:
35-38 l of each sample
For gels to be stained with In-
staStain Ethidium bromide:
18-20 l of each sample

4
.
5

c
m
B

S
e
r
i
e
s

4
8
0

l
C

S
e
r
i
e
s

9
6
0

l
E
x
p
t
.

K
i
t

2
4
0

l
1.5 cm tube
Approximate
Volume
Measurements
1
.
3

c
m
1
.
9

c
m
2
.
8

c
m
Custom bulk quantities are
also available by request.
5. If students have difculty retrieving the entire aliquoted volume of sample because
some of it clings to the side walls of the tubes, remind students to make sure all of
the sample is at the bottom of the tube before gel loading. They should centrifuge
the samples tubes, or tap the tubes on the tabletop.
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Experiment Results and Analysis
In the idealized schematic, the rela-
tive positions of DNA fragments are
shown but are not depicted to scale.
ANALYSIS
Lanes 1 and 2 (set one) represent the crime scene DNA digested by two
different restriction enzymes, which yield distinctly different DNA band-
ing patterns.
Lanes 3 and 4 (set two) represent DNA from Suspect 1. The suspect's
DNA has been digested with the same two restriction enzymes as in
Lanes 1 and 2.
Lanes 5 and 6 (set three) represent DNA from Suspect 2. It also has been
digested with the same two enzymes as the crime scene DNA (Lanes 1
and 2) and DNA from Suspect 1 (Lanes 3 and 4).
Discussion Question:
Could these DNA samples have been distinguished from one another if only
Enzyme 1 had been used?
Lane Tube
1 A DNA from crime scene cut with Enzyme 1
2 B DNA from crime scene cut with Enzyme 2
3 C DNA from Suspect 1 cut with Enzyme 1
4 D DNA from Suspect 1 cut with Enzyme 2
5 E DNA from Suspect 2 cut with Enzyme 1
6 F DNA from Suspect 2 cut with Enzyme 2
( - )
( + )
1 2 3 4 5 6
Please refer to the kit
insert for the Answers to
Study Questions
23
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Experiment
EVT 100202AM
A 0.8 % Agarose Gel Electrophoresis Reference Tables
For DNA Staining with FlashBlue or InstaStain Blue
B 0.8% Agarose Gel Electrophoresis Reference Tables
For DNA Staining with InstaStain Ethidium Bromide
C Quantity Preparations for Agarose Gel Electrophoresis
D Agarose Gel Preparation Step by Step Guidelines
E Staining and Visualization of DNA
FlashBlue liquid
F Staining and Visualization of DNA
InstaStain Blue One-step Staining and destaining
G Staining and Visualization of DNA
InstaStain Blue Cards
H Staining and Visualization of DNA
InstaStain Ethidium Bromide Cards
Appendices
24
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Experiment
Appendix
0.8% Agarose Gel Electrophoresis Reference Tables
(DNA Staining with FlashBlue or InstaStain Blue)
Time and Voltage recommendations for EDVOTEK
equipment are outlined in Table C.1 for 0.8%
agarose gels. The time for electrophoresis will
vary from approximately 15 minutes to 2 hours
depending upon various factors. Conduct the
electrophoresis for the length of time determined
by your instructor.
A
Amt of
Agarose
(g)
Concentrated
Buffer (50x)
(ml)
Size of Gel
(cm)
Distilled
Water
(ml)
Total
Volume
(ml)
7 x 7
7 x 10
7 x 14
0.23
0.39
0.46
0.6
1.0
1.2
29.4
49.0
58.8
+
= +
DNA Staining with FlashBlue or InstaStain Blue
Table
A.1
30
50
60
Table
A.2
Amt of
Agarose
(g)
Diluted
Buffer (1x)
(ml)
Size of Gel
(cm)
7 x 7
7 x 10
7 x 14
0.23
0.39
0.46
30
50
60
+
Individual 0.8%*
DNA Staining with FlashBlue
or InstaStain Blue
For DNA analysis, the recommended
electrophoresis buffer is Tris-acetate-
EDTA, pH 7.8. The formula for
diluting EDVOTEK (50x) concentrated
buffer is one volume of buffer concen-
trate to every 49 volumes of distilled
or deionized water. Prepare buffer as
required for your electrophoresis unit.
50x Conc.
Buffer (ml)
Distilled
Water (ml)
+
EDVOTEK
Model #
Total Volume
Required (ml)
Electrophoresis (Chamber) Buffer
M6+
M12
M36
300
400
1000
Dilution
Table
B
6
8
20
294
392
980
Time and Voltage Guidelines
(0.8% Gel)
Minimum / Maximum Volts
150
125
70
50
15 / 20 min
20 / 30 min
35 / 45 min
50 / 80 min
Table
C.1
M6+ M12 & M36
Minimum / Maximum
25 / 35 min
35 / 45 min
60 / 90 min
95 / 130 min
25
109 Experiment
Appendix
0.8% Agarose Gel Electrophoresis Reference Tables
(DNA Staining with InstaStain Ethidium Bromide)
B
Amt of
Agarose
(g)
Concentrated
Buffer (50x)
(ml)
Size of Gel
(cm)
Distilled
Water
(ml)
Total
Volume
(ml)
7 x 7
7 x 10
7 x 14
0.15
0.23
0.31
0.4
0.6
0.8
19.6
29.4
39.2
+
= +
DNA Staining with InstaStain Ethidium Bromide
Table
A.3
20
30
40
Table
A.4
Amt of
Agarose
(g)
Diluted
Buffer (1x)
(ml)
Size of Gel
(cm)
7 x 7
7 x 10
7 x 14
0.15
0.23
0.31
20
30
40
+
Individual 0.8%*
DNA Staining with InstaStain
Ethidium Bromide
Time and Voltage recommendations for EDVOTEK
equipment are outlined in Table C.1 for 0.8%
agarose gels. The time for electrophoresis will
vary from approximately 15 minutes to 2 hours
depending upon various factors. Conduct the
electrophoresis for the length of time determined
by your instructor.
For DNA analysis, the recommended
electrophoresis buffer is Tris-acetate-
EDTA, pH 7.8. The formula for
diluting EDVOTEK (50x) concentrated
buffer is one volume of buffer concen-
trate to every 49 volumes of distilled
or deionized water. Prepare buffer as
required for your electrophoresis unit.
50x Conc.
Buffer (ml)
Distilled
Water (ml)
+
EDVOTEK
Model #
Total Volume
Required (ml)
Electrophoresis (Chamber) Buffer
M6+
M12
M36
300
400
1000
Dilution
Table
B
6
8
20
294
392
980
Time and Voltage Guidelines
(0.8% Gel)
Minimum / Maximum Volts
150
125
70
50
15 / 20 min
20 / 30 min
35 / 45 min
50 / 80 min
Table
C.1
M6+ M12 & M36
Minimum / Maximum
25 / 35 min
35 / 45 min
60 / 90 min
95 / 130 min
26
109
Experiment
Appendix
C
To save time, the electrophoresis buffer and agarose gel solution can be prepared in
larger quantities for sharing by the class. Unused diluted buffer can be used at a later
time and solidied agarose gel solution can be remelted.
Bulk Electrophoresis Buffer
Quantity (bulk) preparation for 3 liters of 1x electro-
phoresis buffer is outlined in Table D.
Batch Agarose Gels (0.8%)
For quantity (batch) preparation of 0.8% agarose gels,
see Table E.1.
1. Use a 500 ml ask to prepare the diluted gel buf-
fer
2. Pour 3.0 grams of UltraSpec-Agarose into the
prepared buffer. Swirl to disperse clumps.
3. With a marking pen, indicate the level of solution
volume on the outside of the ask.
4. Heat the agarose solution as outlined previously
for individual gel preparation. The heating time
will require adjustment due to the larger total
volume of gel buffer solution.
5. Cool the agarose solution to 60C
with swirling to promote even dis-
sipation of heat. If evaporation
has occurred, add distilled water to
bring the solution up to the original
volume as marked on the ask in
step 3.
60C
Note: The UltraSpec-Agarose kit component is
often labeled with the amount it contains. In many
cases, the entire contents of the bottle is 3.0 grams.
Please read the label carefully. If the amount of
agarose is not specied or if the bottle's plastic seal
has been broken, weigh the agarose to ensure you
are using the correct amount.
Concentrated
Buffer (50x)
(ml)
Distilled
Water
(ml)
Total
Volume
(ml)
60 2,940 3000 (3 L)
= +
Bulk Preparation of
Electrophoresis Buffer
Table
D
3.0 7.5 382.5 390

Batch Preparation of
0.8% UltraSpec-Agarose
Amt of
Agarose
(g)
Concentrated
Buffer (50X)
(ml)
+
Distilled
Water
(ml)
Total
Volume
(ml)
= +
Table
E.1
6. Dispense the required volume of cooled agarose solution for casting each gel. The
volume required is dependent upon the size of the gel bed and DNA staining method
which will be used. Refer to Appendix A or B for guidelines.
7. Allow the gel to completely solidify. It will become rm and cool to the touch after
approximately 20 minutes. Then proceed with preparing the gel for electrophoresis.
Quantity Preparations
for Agarose Gel Electrophoresis
27
109 Experiment
Appendix
EDVOTEK electrophoresis units include
7 x 7 cm or 7 x 14 cm gel casting trays.
A. Using Rubber dams:
Place a rubber dam on each end of the bed. Make sure the rubber dam ts
rmly in contact with the sides and bottom of the bed.
B. Taping with labeling or masking tape:

Extend 3/4 inch wide tape over the sides and bottom edge of the bed.
Fold the extended tape edges back onto the sides and bottom. Press contact
points rmly to form a good seal.
If gel trays and rubber end
caps are new, they may be
initially somewhat difcult to
assemble. Here is a helpful
hint:
2. Place a well-former template (comb) in the rst set of
notches at the end of the bed. Make sure the comb sits
rmly and evenly across the bed.
Preparing the Gel bed
1. Close off the open ends of a clean and dry gel bed (casting tray)
by using rubber dams or tape.
D
Agarose Gel Preparation - Step by Step Guidelines
Place one of the black end caps
with the wide u shaped slot fac-
ing up on the lab bench.
Push one of the corners of the gel
tray into one of the ends of the
black cap. Press down on the tray
at an angle, working from one end
to the other until the end of the
tray completely ts into the black
cap. Repeat the process with the
other end of the gel tray and the
other black end cap.
Casting Agarose Gels
3. Use a 250 ml ask or beaker to prepare the gel solution.
4. Refer to the appropriate Reference Table (i.e. 0.8%, 1.0% or 2.0%)
for agarose gel preparation. Add the specied amount of agarose
powder and buffer. Swirl the mixture to disperse clumps of agarose
powder.
5. With a lab marking pen, indicate the level of the solution volume on
the outside of the ask.
At high altitudes, use
a microwave oven
to reach boiling
temperatures.
6. Heat the mixture to dissolve the agarose powder.
A. Microwave method:
Cover the ask with plastic wrap to
minimize evaporation.
Heat the mixture on High for 1 minute.
Swirl the mixture and heat on High in bursts of 25 seconds
until all the agarose is completely dissolved.
B. Hot plate method:
Cover the ask with aluminum foil to minimize evaporation.
Heat the mixture to boiling over a burner with occasional
swirling. Boil until all the agarose is completely dissolved.
Continue heating until the nal solution appears clear (like water) with-
out any undissolved particles. Check the solution carefully. If you see
"crystal" particles, the agarose is not completely dissolved.
28
109
Experiment
Appendix
7. Cool the agarose solution to 60C with careful swirling to promote
even dissipation of heat. If detectable evaporation has occurred,
add distilled water to bring the solution up to the original volume
marked in step 5.
After the gel is cooled to 60C:
If you are using rubber dams, go to step 9.
If you are using tape, continue with step 8.
DO NOT
POUR
BOILING
HOT
AGAROSE
INTO THE
GEL BED.
Hot agarose solution
may irreversibly warp
the bed.
60C
+
Black Red
Sample wells
During electrophoresis, the

DNA samples migrate through
the agarose gel towards the
positive electrode.
Agarose Gel Preparation
Step by Step Guidelines, continued
D
8. Seal the interface of the gel bed and tape to prevent aga-
rose solution from leaking.
Use a transfer pipet to deposit a small amount of the
cooled agarose to both inside ends of the bed.
Wait approximately 1 minute for the agarose to solidify.
9. Place the bed on a level surface and pour the cooled agarose
solution into the bed.
10. Allow the gel to completely solidify. It will become rm and cool to the touch after approximately
20 minutes.
Preparing the gel for electrophoresis
11. After the gel is completely solidied, carefully and slowly remove the rubber dams or tape from
the gel bed. Be especially careful not to damage or tear the gel wells when removing the rubber
dams. A thin plastic knife, spatula or pipet tip can be inserted between the gel and the dams to
break possible surface tension.

12. Remove the comb by slowly pulling straight up.
Do this carefully and evenly to prevent tearing the
sample wells.
13. Place the gel (on its bed) into the electrophoresis
chamber, properly oriented, centered and level on
the platform.
14. Fill the electrophoresis apparatus chamber with the appropriate amount
of diluted (1x) electrophoresis buffer (refer to Table B on the Appendix
page provided by your instructor).
15. Make sure that the gel is completely submerged under buffer before
proceeding to loading the samples and conducting electrophoresis.
29
109 Experiment
Appendix
E
Staining and Visualization of DNA
FlashBlue Liquid Stain
Preparation of FlashBlue Stain
from Concentrated Solution
Dilute 10 ml of 10x FlashBlue with 90 ml of distilled or
deionized water in a ask. Mix well.
Cover the ask and store it at room temperature until ready
for gel staining.
Do not stain gel(s) in the electrophoresis apparatus.
Staining and Destaining
1. Remove the agarose gel from its bed and and completely submerse the
gel in a small, clean weighboat or lid from pipet tip rack containing
75 ml of 1x FlashBlue stain. Add additional stain if needed to com-
pletely submerge the gel.
2. Stain the gel for 5 minutes.
Note: Staining the gel for longer than 5 minutes will necessitate an extended
destaining time. Frequent changes of distilled water will expedite the process.
Wear Gloves
and Goggles
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. To optimize visibility, use the amber lter
provided with EDVOTEK equipment.
3. Transfer the gel to another small tray and ll it with 250 - 300 ml of
distilled water.
4. Gently agitate the tray every few minutes. Alternatively, place it on a
shaking platform.
Storage and Disposal of FlashBlue Stain and Gel
Gels stained with FlashBlue 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.
Stained gels which are not kept can be discarded in solid waste disposal. FlashBlue stain and
destaining solutions can be disposed down the drain.
5 minute
Staining
30
109
Experiment
Appendix
F
One-Step Staining and Destaining
with InstaStain Blue
1. Remove the 7 x 7 cm agarose gel from its bed and com-
pletely submerse the gel in a small, clean tray containing
75 ml of distilled or deionized water, or used electrophore-
sis buffer. The agarose gel should be completely covered
with liquid.
Examples of small trays include large weigh boats, or small
plastic food containers
InstaStain
One Step
Stain and
Destain
2. Wearing gloves, gently oat a 7 x 7 cm card of InstaStain Blue with the stain side (blue) facing
the liquid.
Note: If staining a 7 x 14 cm gel, use two InstaStain Blue cards.
3. Let the gel soak undisturbed in the liquid for approximately 3 hours. The gel can be left in the
liquid overnight (cover with plastic wrap to prevent evaporation).
4. After staining and destaining, the gel is ready for visualization and photography.
Storage and Disposal of 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.
Wear Gloves
and Goggles
Agarose gels can be stained and destained in one easy step with
InstaStain Blue cards. This one-step method can be completed in
approximately 3 hours, or can be left overnight.
31
109 Experiment
Appendix
1. After electrophoresis, place the agarose gel on
a at surface covered with plastic wrap.
2. Wearing gloves, place the blue dye side of the
InstaStain Blue card(s) on the gel.
3. Firmly run your ngers several times over the
entire surface of the InstaStain card to es-
tablish good contact between the InstaStain
card and the gel.
InstaStain

Patents Pending
DNA InstaStain
Patents Pending
Patents Pending
InstaStain
-
-
-
-
-
1
2
3
4
5
6
Place gel on a flat
surface covered
with plastic wrap.
Place the InstaStain
card on the gel.
Place a small weight
for approx. 5 minutes.
Transfer to a small
tray for destaining.
Destain with 37C
distilled water.
Press firmly.
InstaStain is a registered trademark of EDVOTEK, Inc. Patents Pending.
Instastain Blue Cards
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.
5. Allow the InstaStain Blue to sit on the gel for 5 to 10 minutes.
6. After staining, remove the InstaStain card.
If the color of the gel appears very light, wet the gel surface with buffer or
distilled water and place the InstaStain card on the gel for an additional 5
minutes.
Destaining and Visualization of DNA
7. Transfer the gel to a large weigh boat or small plastic container.
8. Destain with approximately 100 ml of distilled water to cover the gel.
9. Repeat destaining by changing the distilled water as needed.
Larger DNA bands will initially be visible as dark blue bands against a lighter
blue background. When the gel is completely destained, larger DNA bands
will become sharper and smaller bands will be visible. With additional de-
staining, the entire background will become uniformly light blue. Destaining
time may vary between 20 - 90 minutes.
10. Carefully remove the gel from the destain solution and examine the gel
on a Visible Light Gel Visualization System. To optimize visibility, use the
amber lter provided with EDVOTEK equipment.
11. If the gel is too light and bands are difcult to see, repeat the staining
and destaining procedures.
Wear Gloves
and Goggles
G
32
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Experiment
Appendix
Instastain Blue Cards
continued
Destaining Notes for InstaStain Blue
Use of warmed distilled water at 37C will accelerate destaining. Destaining will take longer with
room temperature water.
DO NOT EXCEED 37C ! 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 small-
est 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 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.
G
33
109 Experiment
Appendix
1. After electrophoresis, place the gel on a piece
of plastic wrap on a at surface. Moisten the
gel with a few drops of electrophoresis buffer.
2. Wearing gloves, remove the clear plastic pro-
tective sheet, and place the unprinted side of
the InstaStain EtBr card(s) on the gel.
Disposal of InstaStain
Disposal of InstaStain cards and gels should follow institutional
guidelines for chemical waste.
Additional Notes About Staining
If bands appear faint, or if you are not using EDVOTEK UltraSpec-
Agarose, gels may take longer to stain with InstaStain EtBr.
Repeat staining and increase the staining time an additional 10-15
minutes.
DNA markers should be visible after staining even if other DNA
samples are faint or absent. If markers are not visible, troubleshoot
for problems with the electrophoretic separation.
Caution: Ethidium Bromide is a listed mutagen.
InstaStain Ethidium Bromide Cards
3. Firmly run your ngers over the entire surface of the InstaStain EtBr.
Do this several times.
4. Place the gel casting tray and a small empty beaker on top to ensure
that the InstaStain card maintains direct contact with the gel surface.
Allow the InstaStain EtBr card to stain the gel for 3-5 minutes.
5. After 10-15 minutes, remove the InstaStain EtBr card. Transfer the gel
to a ultraviolet (300 nm) transilluminator for viewing. Be sure to wear
UV protective goggles.
Wear Gloves
and Goggles
H
1
2
3
4
5
Press firmly.
Moisten
the gel.
Place the InstaStain
card on the gel.
Place a small weight to
ensure good contact.
View on U.V. (300 nm)
transilluminator

InstaStain
Patents Pending
DNA InstaStain
Patents Pending
Patents Pending
InstaStain
-
-
-
-
-
Material Safety Data Sheets
Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
109
Experiment
34
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35
109
Experiment
Material Safety Data Sheets
Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
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s
The Biotechnology Education Company
EDVOTEK, Inc. 1-800-EDVOTEK vvv.edvotek.com
EDVOTEK Series 100 Electrophoresis Experiments:
Cat. # Title
101 Principles and Practice of Agarose Gel Electrophoresis
102 Restriction Enzyme Cleavage of Plasmid & Lambda DNA
103 Principles of PCR
104 Size Determination of DNA Restriction Fragments
105 Mapping of Restriction Sites on Plasmid DNA
109 DNA Fingerprinting by Restriction Enzyme Patterns
112 Restriction Enzyme Cleavage of Lambda DNA
114 DNA Paternity Testing Simulation
115 Cancer Gene Detection
116 Sickle Cell Gene Detection (DNA-based)
117 Detection of Mad Cow Disease
118 Cholesterol Diagnostiics
124 DNA Screening for Smallpox
130 DNA Fingerprinting by PCR Amplification

DNA Fingerprinting

Diunggah oleh

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The Biotechnology Education Company

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

Agarose Gel Electrophoresis

During electrophoresis, the

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