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


EVT 2011_08_22AM
EDVO-Kit #
962
Identication of
Foodstuffs from
Genetically Modied
Organisms
Storage: See Page 3 for
specic storage instructions
EXPERIMENT OBJECTIVE:
The objective of this experiment is to utilize PCR
to identify genetically modied foods.
This experiment is designed for DNA staining with InstaStain Ethidium Bromide.
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962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
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.
Experiment Components 3
Experiment Requirements 4
Background Information 5
Experiment Procedures
Experiment Overview and General Instructions 10
Module I: Isolation of DNA from Food 12
Module II: PCR Amplication 13
Module III: Separation of PCR Reactions by
Agarose Gel Electrophoresis 15
Study Questions 16

Instructors Guidelines
Notes to the Instructor 17
Pre-Lab Preparations 21
Experiment Results and Analysis 22
Study Questions and Answers 23
Appendices
A PCR Success Guidelines 26
B Polymerase Chain Reaction Using Three Waterbaths 27
C Preparation and Handling of PCR Samples With Wax 28
D 2.0% Agarose Gel Electrophoresis Reference Tables 29
E Buffer and Agarose Quantity Preparations 30
F Agarose Gel Preparation 31
G Staining and Visualization of DNA with InstaStain 33
Ethidium Bromide Cards
H Staining and Visualization of DNA - FlashBlue Liquid Stain 34
I InstaStain Blue: One Step Staining and Destaining 35
J Electrophoresis Hints and Help 36
Material Safety Data Sheets 38
Table of Contents
3
962
Experiment #
EVT 2011_08_22AM
EDVOTEK - The Biotechnology Education Company
1-800-EDVOTEK www.edvotek.com
FAX: (301) 340-0582 email: info@edvotek.com
Identication of Foodstuffs
from Genetically Modied Organisms
Experiment Components
All components are
intended for educational
research only. They are not
to be used for diagnostic or
drug purposes, nor admin-
istered to or consumed by
humans or animals.
THIS EXPERIMENT DOES
NOT CONTAIN HUMAN
DNA. None of the experi-
ment components are de-
rived from human sources.

Storage
A Tubes with PCR reaction pellets Room Temperature
Each PCR reaction pellet contains
dNTP Mixture
Taq DNA Polymerase Buffer
Taq DNA Polymerase
MgCl
2
B GMO Primer mix -20C Freezer
C 100 base pair ladder -20C Freezer
D Positive PCR control -20C Freezer
E Tris-EDTA Buffer -20C Freezer
F Proteinase K Room temperature
G NaCl Room temperature
H DNA extraction buffer Room temperature
Reagents & Supplies
(Store all components below at room temperature)
UltraSpec-Agarose
Electrophoresis Buffer (50x)
10x Gel Loading Solution
InstaStain Ethidium Bromide
InstaStain Blue
Microcentrifuge Tubes
PCR tubes (0.2 ml - for thermal cyclers with 0.2 ml template)
Calibrated transfer pipets
Wax beads (for waterbath option or thermal cyclers without heated lid)
This experiment is
designed for 10 lab
groups.
Sample volumes are very
small. For liquid samples,
it is important to quick
spin the tube contents in a
microcentrifuge to obtain
sufcient volume for pipet-
ing. Spin samples for 10-20
seconds at maximum speed.
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962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Experiment Requirements
Recommended foodstuffs that have worked well in the EDVOTEK
testing laboratory include: corn akes, soybeans, cornmeal, corn chips,
soy snacks.
Thermal cycler (EDVOTEK Cat. # 541 highly recommended)
or three waterbaths*
Horizontal gel electrophoresis apparatus
D.C. power supply
Balance
Microcentrifuge
Water bath (56C)
UV Transilluminator or UV Photodocumentation system
UV safety goggles
Automatic micropipets (5-50 l) with tips
Microwave, hot plate or burner
Pipet pump
250 ml asks or beakers
Hot gloves
Disposable vinyl or latex laboratory gloves
Ice buckets and ice
Distilled or deionized water
Isopropanol
Online Ordering
now available
Visit our web site for information
about EDVOTEKs complete line
of hands-on experiments for
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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
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*If you do not have a thermal
cycler, PCR experiments can
be conducted, with proper
care, using three waterbaths.
However, a thermal cycler
assures a signicantly higher
rate of success.
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document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
Copyright 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
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962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Background Information
Tomatoes, soybeans, and corn were among the rst genetically modied food
products approved by U.S. agencies in the 1990s. Since then food biotechnology
continues to grow rapidly. Enormous advances in genetics over the last decade
have led to accelerated changes in biotechnology, with ramications in social and
political elds. Genetic research which ushered our understanding about the
very basis of life also led the heated debates on what should be done in the area
of genetically modied (GM) foods and what may be possible in the future.
Debates on GM foods are world-encompassing, with arguments citing risks and
benets for crops in Brazil, countries in Africa, Europe, Bolivia, India, Japan,
Australia and New Zealand, as well as the United States. Articles on the topic
have been published in leading science journals, including Scientic American,
Nature and Science, and there have been impassioned discussions in government
chambers and in environmental, religious and ethical circles. On one side of the
controversy are the promises of improved quantity and quality plants, decreasing
costs for growers, and benets to the environment; on the other side are fears
of damage to the environment and to other crops, increased allergens, and the
creation of other unanticipated dangers to people and the environment.
Adaptation is vital to all living things in nature and is the key to evolution. Na-
ture is dynamic and at all times is changing and as a consequence adaptation to
change becomes a prerequisite to survival. In one sense plant genetics is not new
to man. Over the centuries selective breeding and conventional hybridization
provided mankind a distinct advantage over natural selection. Biotechnology
has redirected and accelerated the pace of selective breeding and evolution and
introduced possibilities which until recently were not considered to be feasible.
A goal of plant genetics is the development of plants that yield optimum product
and have selective advantages. With the advent of biotechnology, cloning and
expression of genes in GM plants have increased yields, nutritional value and en-
hanced quality. Plant biotechnology today offers the possibilities of modication,
enhancement or suppression of gene products.
In the last half of the century, the world population more than doubled however
agriculture only increased by 10%. In the same time frame world food produc-
tion per person increased by 25% due to advances in agriculture due to mecha-
nization and biotechnology. For example, in 2002, 74% (80 million acres) of
American soybeans were obtained from genetically-modied crops. The benets
of food production have not been equally distributed amongst the world popula-
tion with the U.S. being both the largest producer and consumer of food.
APPROACHES TO PLANT BIOTECHNOLOGY
Introduction of specic genes through biotechnology can provide advantages. As
an example, a genetically modied (GM) plant can protect itself against parasites
after the introduction of the endotoxin gene. Golden rice is an example of a
GM crop that synthesizes a high value bioproduct. Plants can also be modied to
inhibit the expression of specic genes that are involved in the ripening of fruits
by maintaining and enhancing fruit avors and extending their shelf life.
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962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Background Information
There are various biotechnology procedures that can be used in plant genetic en-
gineering. Examples include the use of gene guns, Ti plasmid based gene intro-
duction and antisense technology. The gene gun approach uses tiny metal beads
coated with the specic DNA that is targeted to be introduced in plant cells. The
pellets are shot into embryonic plant cells, and treated cells are screened for the
gene of interest through the use of specic markers. An example of the use of
the gene gun for the production of a recombinant plant is the Bt corn. Genes
isolated from Bacillus thuringiensis together with the CaMV/35S promoter are
red and enter the cells resulting in the expression of an endotoxin. The bacte-
rial endotoxin serves as a pesticide for the corn plant. Parasitic insects that feed
on the corn plant ingest the bacterial endotoxin which causes a break down of
their gut wall and results in eventual death.
The Ti plasmid from the soil bacterium Agrobacterium tumefaciens is used as a
vector for transferring DNA into dicotyledonous plants. These plants have two
seed halves such as tomatoes, apple and soybean. Monocotyledonous plants,
grow from a single seed embryo such as corn and wheat and are genetically ma-
nipulated by different technologies.
In some cases, antisense technology is employed for improving plant products.
The Flavr Savr tomatoes contain an anti-complementary copy of the gene that
codes for the enzyme polygalacturonase (PG). The antisense mRNA binds and
inactivates the normal mRNA, thus inhibiting translation and shutting down the
production of PG. As a result pectin is not digested and ripening is slowed.
Golden rice, named for its appearance, is genetically modied to produce precur-
sor metabolites of vitamin A. This vitamin is essential for nutrition and is chroni-
cally decient in diets in developing countries. Rice normally lacks beta carotene
but contains geranyl diphosphate a (precursor of -carotene and of vitamin A).
Rice is bombarded with cDNA from bacteria that contain the phyteone synthase
gene. This enzyme condenses two C20 to C40, which after several enzymatic
steps, is converted to -carotene (C40). Upon ingestion in the presence of fat, the
pro-vitamin -carotene is converted into Vitamin A. Delivery of a vitamin in rice
(which is the staple food for a large segment of the world population) is a major
step forward in supplementing food with key high value targeted nutritional
components.
A future approach to food Agro-biotechnology will include the introduction
of genes in chloroplasts which can accept several different genes. Chloroplast
genetic engineering is rapidly becoming a viable focus for researchers. Like mi-
tochondria, chloroplast genes typically have high expression, and are not passed
through the pollen. With high protein expression levels and little chance of non-
target exposure to the product, chloroplast engineering has made plant-based
expression of pharmaceuticals a real possibility. Biopharming to produce Farma-
ceuticals has the potential to use any number of crops such as tobacco, carrots,
tomatoes, soybeans and rice for their plant-made pharmaceuticals.
The responsibility of public health and policy concerning agro-biotechnology
rests on the shoulders of both the public and the biotechnology industry. It
remains to be seen what long-term effects altered plants will have on the eco-
system and overall biodiversity. To gain acceptance, the plant biotechnology
7
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document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
Copyright 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Background Information
industry needs to better communicate its research and development of new GM
foods products. In the future one can foresee allergy free peanuts, low protein
rice that would help kidney disease compromised individuals, low fat and high
protein GM foods. Genetically modied foods may also be foreseen to serve as
vehicles for the delivery of various pharmaceuticals.
There are several Federal agencies in the United States that oversee various
aspects of food safety. The Federal Drug Administration (FDA) is responsible for
the safety of food and animal feed products. The U.S. Department of Agriculture
(USDA) oversees new plant varieties that are safe for the environment. Lastly,
the Environmental Protection Agency (EPA) monitors and sets standards for pes-
ticide levels in plants and determines what is acceptable for human consumption.
ABOUT THE POLYMERASE CHAIN REACTION
The polymerase chain reaction (PCR) is a relatively new tool for DNA amplica-
tion that has revolutionized almost all aspects of biological research. PCR was
invented in 1984 by Dr. Kary Mullis while at Cetus Corporation. Mullis was
awarded a Nobel Prize for his work in 1994. PCR allows for the amplication of a
small quantity of DNA over one million-fold in a few hours. The enormous utility
of PCR is based on its procedural simplicity and specicity. Since the rst appli-
cation of PCR to diagnose sickle cell anemia, a large number of diagnostic tests
have been developed. Many such diagnostic tests have now become routine.
PCR has also made amplication of DNA an alternate approach to cloning experi-
ments. It is used extensively in plant agrobiotechnology as well as various other
elds of biotechnology. For example biomedical applications include diagnostic
test for infectious agents and inherited or acquired genetic conditions including
various forms of cancer.
PCR amplication requires the use of a thermostable DNA polymerase. Taq poly-
merase is the most commonly used polymerase which is puried from a bacteri-
um known as Thermus Aquaticus that inhabits hot springs. This enzyme remains
stable at near-boiling (95C) temperatures. Also required in the PCR reaction
are the four deoxynucleotides (dATP, dCTP, dGTP, and dTTP) and two synthetic
oligonucleotides, typically 15-30 base pairs in length, known as primers. These
components, together with the DNA to be amplied, are incubated in an appro-
priate buffer that contains Mg2+. The primers are designed to correspond to the
start and end of the amplied sequence, known as the template or target.
If the template DNA is prepared from biological tissue, freshly isolated DNA will
give the best amplication results. DNA extracted from older specimens may be
degraded and be less suitable for amplication.
The PCR reaction mixture that contains Taq polymerase, buffer, the four de-
oxytriphosphate nucleotides, primers, and template is sequentially subjected to
three dened temperatures. The reaction mixture is held at each temperature
step for dened amounts of time that usually range from 30 to 60 seconds.
In the rst step, the reaction mixture is heated to near boiling (94 - 96C) to
denature or melt the DNA. This step, known as denaturation disrupts the
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962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
hydrogen bonds between the two complementary DNA strands and causes their
separation. In the second PCR step, the mixture is cooled to a temperature that
is typically in the range of 45 65. In this step, known as annealing, the
primers, present in great excess of the template, bind to the separated DNA
strands. In the third PCR step, known as extension, the temperature is raised
to an intermediate value, usually 72C. At this temperature the Taq polymerase
is maximally active and adds nucleotides to the primers to complete the synthesis
of the new complementary strands.
The exact temperature and incubation time required at each step depends on
several factors, including the length and sequence of the DNA to be amplied
the length and GC content of the primers, and the primer/template ratio.
The three PCR steps of denaturation, annealing, and extension constitute one
cycle and result in a doubling of the number of copies of the template to be
amplied. The process is typically repeated for 20-40 cycles. Theoretically, if the
reaction is subjected to 30 PCR cycles, the anticipated number of DNA templates
copies will be over a million copies. The amplied DNA is then subjected to aga-
rose gel electrophoresis for analysis.
Background Information
9
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 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
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3' 5'
3' 5'
5' 3'
5' 3'
5'
5' 3'
3' 5'
5' 3'
5'
5'
Denature
94C
5'
Extension
72C
3' 5'
Separation of
two DNA strands
=
Primer 1 =
Primer 2 =
5' 3'
5'
Anneal
2 primers
40C - 65C
3' 5'
5'
5'
5'
3' 5'
5'
5'
5' 3'
5'
5'
5'
5' 3'
5' 3'
5' 3'
5' 3'
5' 3'
5' 3'
5'
5' 3'
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Target Sequence
5' 3'
5' 3'
5' 3'
Background Information
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from Genetically Modied Organisms
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Experiment Overview and General Instructions
BEFORE YOU START THE EXPERIMENT
1. Read all instructions before starting the experiment.
2. If you will be conducting PCR using a thermal cycler without a heated lid,
also read the Appendix entitled "Preparation and Handling PCR Samples
with Wax ".
3. If you will be using three waterbaths to conduct PCR, read the two appen-
dices entitled "Polymerase Chain Reaction Using Three Waterbaths" and
"Handling samples with wax overlays".
4. Write a hypothesis that reects the experiment and predict experimental
outcomes.
EXPERIMENT OBJECTIVE:
The objective of this experiment is to utilize PCR to identify genetically modied
foods.

BRIEF DESCRIPTION OF EXPERIMENT:
In this experiment, students will identify food that may contain the CaMV 35S
promoter region, the NOS terminator, and/or the plant chloroplast gene. Am-
plication of the PCR products will be performed and analyzed by agarose gel
electrophoresis.
Note:
Not all foods will consist of GMO material and may result in negative PCR products.
Also, the quality of DNA obtained from processed food will vary widely. Conse-
quently, positive PCR results (bands) are not guaranteed. Foods that have worked
well in the EDVOTEK testing laboratory include corn akes, soy crisps and corn
chips.
GEL SPECIFICATIONS
This experiment requires a gel with the following specications:

Recommended gel size 7 x 14 cm (long tray)
Number of sample wells required 6
Placement of well-former template rst set of notches
Gel concentration required 2.0%
11
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 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
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Experiment Overview and General Instructions
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.
Wear gloves
and safety goggles
4. Exercise caution when using any electrical equipment in the laboratory.
Although electrical current from the power source is automatically
disrupted when the cover is removed from the apparatus, rst turn
off the power, then unplug the power source before disconnecting
the leads and removing the cover.
Turn off power and unplug the equipment when not in use.
5. EDVOTEK injection-molded electrophoresis units do not have glued junc-
tions that can develop potential leaks. However, in the unlikely event
that a leak develops in any electrophoresis apparatus you are using, IM-
MEDIATELY SHUT OFF POWER. Do not use the apparatus.
6. Always wash hands thoroughly with soap
and water after handling reagents or
biological materials in the laboratory.
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Module I: Isolation of DNA from Food
1. Weigh 50-100 mg of food and transfer it into a test tube.
2. Add 400 l of extraction buffer to the tube.
3. Mash the food material well with a micropestle.
4. Incubate the tube at 56C for at least one hour, or overnight.
5. Add 300 l of NaCl solution to the tube.
6. Mix well for 30 seconds by vortexing or vigorous tapping with your nger.
7. Centrifuge for 15 - 30 minutes at full speed.
8. Remove the supernatant and transfer it to a fresh tube. Discard the tube
with the pellet.
9. Add an equal volume of 100% isopropanol or 91% isopropyl (rubbing) alco-
hol to the tube containing the supernatant.
10. Incubate the tube in the freezer for at least one hour, or overnight.
OPTIONAL STOPPING POINT - Incubate at 56C overnight.
OPTIONAL STOPPING POINT - Incubate in the freezer overnight.
11. Spin the tube at full speed for 20 minutes.
12. Using care to avoid disturbing the pellet, completely remove and discard the
supernatant from the pellet.
13. Wash the pellet with 1.5 ml of 70% ethanol or isopropanol by slowly adding
the alcohol and then removing it.
14. If the pellet becomes dislodged, spin at full speed for two minutes.
15. Remove and discard the alcohol and allow the pellet to dry completely.
16. Dissolve the DNA pellet in 300 l of 1x TE buffer. Place the DNA sample on
ice or in the freezer until sample preparation for amplication.
OPTIONAL STOPPING POINT - Store in the freezer until ready for PCR.
13
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 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
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Module II: Amplication of DNA
The PCR reaction pel-
let contains Taq DNA
polymerase, the four
deoxytriphosphates, Mg
+2
and buffer.
Sample volumes are very
small. For liquid samples,
it is important to quick
spin the tube contents in
a microcentrifuge to ob-
tain sufcient volume for
pipeting. Spin samples for
10-20 seconds at maxi-
mum speed.
PREPARE SAMPLES FOR POLYMERASE CHAIN REACTION
1. Transfer the PCR Reaction pellet to the appropriate sized tube (e.g.
0.5 ml or 0.2 ml) for your thermal cycler.
2. Tap the PCR tube to assure the PCR reaction pellet is at the bottom of
the tube.
3. Label the tube with the sample and with your initials.
4. To prepare the PCR reaction mix, add the following to the pellet:
Food DNA Template for Amplication 5 l
Primer Mix (two primers) 20 l
5. Gently mix the reaction tube. Make sure the PCR reaction pellet is
completely dissolved.
6. If your thermal cycler is equipped with a heated lid, proceed directly to
polymerase chain reaction cycling.
If your thermal cycler does not have a heated lid, or if you are cycling
manually with three water baths, add one wax bead to the tube before
proceeding to polymerase chain reaction cycling.
Control Reaction:
This kit provides reagents for 21 reactions which includes the control reac-
tion. A control tube "C" (Control) will be assembled to be used for the entire
class or per gel. Your instructor will provide that information to the class. To
assemble the control reaction, add 5 l of control DNA Template for Amplica-
tion and 20 l of Primer Mix to the tube containing the pellet. Place this tube
in the thermal cycler with the samples from the class.
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Experiment
Identication of Foodstuffs
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POLYMERASE CHAIN REACTION CYCLING
7. Each group should place their tube(s) in the thermal cycler (and the optional
control reactions) for automatic cycling as programmed.
Initial Denaturation 50 cycles @ Final Extension
94C for 10 min. 94C for 1 min. 72C for 10 min.
63C for 1 min.
72C for 1 min.
8. Once the PCR process is complete, add 5 l of 10x gel loading solution to
each sample. Store on ice until ready for electrophoresis.
9. Proceed to instructions for preparing a 2.0% agarose gel (7 x 14 cm) and
separating the PCR products by electrophoresis.
OPTIONAL STOPPING POINT
Samples may be placed in the freezer until ready for agarose gel
electrophoresis.
Module II: Amplication of DNA
15
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 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
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962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Module III: Separation of PCR Reactions by Agarose Gel Electrophoresis
PREPARE THE GEL
1. Prepare a 2.0% agarose gel and is designed for staining with InstaStain
Ethidium Bromide. Refer to Appendix D.
Recommended gel size: 7 x 14 cm
7 x 14 cm gels are recommended to achieve better resolution of the PCR products.
Each gel can be shared by several students or groups.
Placement of well-former template: rst set of notches
Agarose gel concentration: 2.0%
LOAD DNA SAMPLES
2. (Optional step) Heat the 100 bp DNA ladder and PCR samples for two min-
utes at 50C. Allow the samples to cool for a few minutes.
3. Make sure the gel is completely submerged under buffer before loading the
samples. Load the DNA ladder in lane 1 of each gel.
4. Load the entire volume (approx. 30 l) of each PCR sample in consecutive
wells.
Remember to note the wells in which your groups samples are loaded.

RUN THE GEL
5. After the DNA samples are loaded, set the power source at the required
voltage and conduct electrophoresis for the length of time specied by your
instructor.
STAIN AND VISUALIZE DNA

6. After the electrophoresis is completed, proceed to DNA staining and visual-
ization (see Appendices G, H, or I for staining instructions).
If you are unfamiliar
with agarose gel prepa-
ration step by step
guidelines are outlined
in Appendix F.
Reminder:
Before loading the samples,
make sure the gel is
properly oriented in the
apparatus chamber.
+ -
Black Red
Sample wells
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Answer the following study questions in your laboratory notebook or on a sepa-
rate worksheet.
1. How are gene guns used in plant genetics? Discuss an example of the pro-
cess.
2. What are common potential health concerns about foodstuffs obtained from
GM plants?
3. Which Federal agencies are responsible for oversight on GM plants and food-
stuffs?
4. How does antisense technology assist in delaying the ripening of fruit and
other plant products?
5. How are primers designed to assure that the entire target is amplied by
PCR?
Study Questions
17
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Identication of Foodstuffs
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Instructors
Guide
Class size, length of laboratory sessions, and availability of equipment are
factors which must be considered in the planning and the implementation
of this experiment with your students. These guidelines can be adapted
to t your specic 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. In addition, Technical Service is available
from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowl-
edgeable technical staff at 1-800-EDVOTEK (1-800-338-6835).
NATIONAL CONTENT AND SKILL STANDARDS
By performing this experiment, students will learn to load samples and
run agarose gel electrophoresis. Analysis of the experiments will provide
students the means to transform an abstract concept into a concrete expla-
nation. Please visit our website for specic content and skill standards for
various experiments.
Notes to the Instructor
Visit our web site for information
about EDVOTEK's complete line
of experiments for biotechnology
and biology education.
EDUCATIONAL RESOURCES
Electrophoresis Hints, Help and Frequently
Asked Questions
EDVOTEK experiments are easy to perform and
designed for maximum success in the classroom
setting. However, even the most experienced
students and teachers occasionally encounter
experimental problems or difculties. The ED-
VOTEK web site provides several suggestions and
reminders for conducting electrophoresis, as well
as answers to frequently asked electrophoresis
questions.
Visit the EDVOTEK web site often for
continuously updated information.
M
o
n
- Fri 9 a
m

-

6

p
m

E
T
(1-800-338-6835)
E
D
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T
E
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: info@edvotek.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
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PCR EXPERIMENTAL SUCCESS GUIDELINES
Please refer to the Appendices section for a summary of important hints and re-
minders which will help maximize successful implementation of this experiment.
This experiment has three modules:
I. Isolation of DNA
II. PCR Amplication of DNA
III. Separation of PCR Reactions by Electrophoresis
MICROPIPETTING BASICS AND PRACTICE GEL LOADING
Accurate pipeting is critical for maximizing successful experiment results. ED-
VOTEK Series 300 experiments are designed for students who have had previous
experience with agarose gel electrophoresis and micropipeting techniques. If
your students are unfamiliar with using micropipets, EDVOTEK highly recom-
mends that students perform Experiment # S-44, Micropipetting Basics, or other
Series 100 or 200 electrophoresis experiment prior to conducting this advanced
level experiment.
APPROXIMATE TIME REQUIREMENTS
1. The PCR step (35 cycles) will take about 150-180 minutes or can be processed
overnight and held at 4C.
2. The experiment can be temporarily stopped after the completion of Module
1 or Module II and later resumed. Experimental results will not be compro-
mised if instructions are followed as noted under the heading Optional
Stopping Point.

3. 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 solidication.
See section Options for Preparing Agarose
Gels below.
4. The approximate time for electrophore-
sis will vary from 40 minutes to 3.5 hours.
Generally, the higher the voltage applied,
the faster the samples migrate. However,
depending upon the apparatus congura-
tion and the distance between the two
electrodes, individual electrophoresis units
will separate DNA at different rates. Follow
manufacturer's recommendations. Time
and Voltage recommendations for EDVOTEK
equipment are outlined in Table C.
Time and Voltage Guidelines
2% UltraSpec-Agarose Gel
Time Volts
125
70
50
40 min
2.0 hrs
3.0 hrs
Approx. migration
distance of
tracking dye
Gel Size (cm)
Volume
7 x 7
25 ml
7 x 14
50 ml
125
70
50
60 min
2.5 hrs
3.5 hrs
4.5 cm
6.0 cm
Table
C
Notes to the Instructor
19
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document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
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OPTIONS FOR PREPARING AGAROSE GELS
This experiment is designed for DNA staining after electrophoresis with InstaStain
Ethidium Bromide. There are several options for preparing agarose gels for the
experiment.
1. Individual Gel Casting:
Each student lab group can be responsible for casting their own individual
gel prior to conducting the experiment.
2. 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.
Gels that have been removed from their trays for storage, should be "an-
chored" 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
gels from sliding around in the trays and the chambers.
3. 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 sharing by
the class. See instructions for "Batch Gel Preparation".
GEL CONCENTRATION AND VOLUME
The gel concentration required for this experiment is 2.0%. Prepare gels accord-
ing to Table A.1 or A.2 in Appendix D.
Notes to the Instructor
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GEL STAINING AND DESTAINING AFTER ELECTROPHORESIS

After electrophoresis, the agarose gels require staining in order to visualize the
separated DNA samples. This experiment features a proprietary stain called
InstaStain.
InstaStain EtBr (Appendix F)
Optimal visualization of PCR products on gels of 1.0% or higher concentration is
obtained by staining with InstaStain Ethidium Bromide (InstaStain EtBr) cards.
Exercise caution when using Ethidium Bromide, which is a listed mutagen. Dispos-
al of the InstaStain EtBr cards, which contain only a few micrograms of ethidi-
um bromide, is minimal compared to the large volume of liquid waste generated
by traditional ethidium bromide staining procedures. Disposal of InstaStain
cards and gels should follow institutional guidelines for chemical waste.
InstaStain Blue: One-step Staining and Destaining (Appendix G)
InstaStain Blue can be used as an alternative for staining gels in this experi-
ment. However, InstaStain Blue is less sensitive than InstaStain EtBr and will
yield variable results.
Agarose gels can be stained and destained in one easy step, which can be com-
pleted in approximately 3 hours, or can be left in liquid overnight. For the best
photographic results, leave the gel in liquid 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.
Gels stained with InstaStain Blue 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 Blue cards and destained gels can be
discarded in solid waste disposal. Destaining solutions can be disposed down the
drain.
PHOTODOCUMENTATION OF DNA (OPTIONAL)
There are many different photodocumentation systems available, including digi-
tal systems that are interfaced directly with computers. Specic instructions will
vary depending upon the type of photodocumentation system you are using.
Notes to the Instructor
21
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 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
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Pre-Lab Preparations
ISOLATION OF DNA
(Components E-H)
If a precipitate has formed in the DNA extraction buffer,
warm at 37C to redissolve.
1. Add 200 l of DNA Extraction Buffer (H) to each
tube of Proteinase K (F) and allow the pellets to
hydrate for a couple of minutes. Add the dissolved
Proteinase K back to the 10 ml of DNA Extraction
Buffer and mix. Aliquot 1 ml for each group and
keep tubes on ice.
2. Aliquot 1 ml of NaCl solution (G) for each group.
Materials for DNA Isolation
Each student group should receive:
1 ml DNA extraction buffer
1 ml NaCl solution
1 ml TE buffer
2 Microcentrifuge tubes with pestles
4 1.5 ml microcentrifuge tubes with caps
Materials for PCR
Each student group
should receive:
50 l GMO Primer Mix
20 l Gel load solution
2 PCR beads
35 l 100 bp ladder
POLYMERASE CHAIN REACTION
(Components A-D)
Aliquot reagents for PCR.
1. GMO Primer Mix (B)
Aliquot 50 l for each group
For the group performing the control reaction, aliquot an addi-
tional 20 l.
2. Aliquot 20 l gel load solution for each group
3. Aliquot 35 l of 100 bp ladder (C) for each student group.
4. Thaw the positive control sample (D) and dispense 7 l for the one
group performing the control. Place on ice.
Notes and Reminders:
Accurate temperatures and cycle times are critical. A pre-run for one cycle (approx. 3 to 5
min) is recommended to check that the thermal cycler is properly programmed.
For thermal cyclers which do not have a top heating plate, it is necessary to place a layer of wax
above the PCR reactions in the microcentrifuge tubes to prevent evaporation. See Appendix
entitled "Preparation and Handling PCR Samples with Wax ".
3. Aliquot 1 ml of TE buffer (E) for each group
4. Place bottles of 95% and 70% Isopropyl alcohol on ice or in the freezer.
Chill thoroughly.
There is enough material for ten student groups sharing ve gels (two groups
per gel). There is material to perform one positive control DNA reaction for PCR.
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Experiment Results and Analysis
* Note:
Depending on the PCR conditions used, a diffuse, small-molecular weight band, known
as a "primer dimer", may be present below the 100 bp marker (shown in the idealized
schematic at the bottom of the gel). This is a PCR artifact and can be ignored. Other
minor bands may also appear due to nonspecic primer binding and the subsequent
amplication of these sequences.
Lane 1 100 bp ladder
Lane 2 GMO marker
(500 bp plant chloroplast, 200 bp CMV 35S
and 127 bp NOS terminator)
Lane 3 GMO food (corn akes)
(Positive for plant chloroplast, CMV 35S
and NOS terminator)
Lane 4 non-GMO food
(Positive for plant chloroplast only)
Lane 5 GMO food (soy chips)
(positive for plant chloroplast, CMV 35S
and NOS Terminator)
500 bp
100 bp
1 2 4 3 5 6
In this experiment, results will vary depending upon the type of genetic modi-
cation (if any) in the food source chosen by the student(s). Successful genomic
DNA purication from foodstuffs can have a signicant impact on the PCR am-
plication and gel electrophoresis results. Poor results and quality of extracted
genomic DNA can be caused by an unsuccessful extraction attempt. For optimal
DNA preparation, particular attention should be paid to the extraction process as
described in the protocol.
*
Please refer to the kit
insert for the Answers to
Study Questions
24
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Experiment
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Notes
25
962
Experiment #
EVT 2011_08_22AM
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FAX: (301) 340-0582 email: info@edvotek.com
Identication of Foodstuffs
from Genetically Modied Organisms
Appendices

A PCR Success Guidelines
B Polymerase Chain Reaction Using Three Waterbaths
C Preparation and Handling of PCR Samples With Wax

D 2.0% Agarose Gel Electrophoresis Reference Tables
E Buffer and Agarose Quantity Preparations
F Agarose Gel Preparation
G Staining and Visualization of DNA with InstaStain Ethidium
Bromide Cards
H Staining and Visualization of DNA - FlashBlue Liquid Stain
I InstaStain Blue: One Step Staining and Destaining
J Electrophoresis Hints and Help
Material Safety Data Sheets
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Experiment
Identication of Foodstuffs
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Appendix
EDVOTEK experiments which involve the amplication of DNA are extremely relevant,
exciting and stimulating classroom laboratory activities. These experiments have been per-
formed successfully in many classrooms across the country, but do require careful execution
because of the small volumes used. The following guidelines offer some important sugges-
tions, reminders and hints for maximizing success.
THE PCR REACTION
1. Add Primers and DNA to the PCR Reaction Bead: Add the primer mixture (forward
and reverse primers) and the cell DNA (supernatant) as specied in the experimental
procedures to the microcentrifuge tube containing the PCR reaction bead. Make sure
that the bead (which contains the Taq DNA polymerase, the 4XdTPs, Mg and the PCR
reaction buffer) is completely dissolved. Do a quick spin in a microcentrifuge to bring
the entire sample to the bottom of the tube. Prepare the control reaction similarly.
2. The Thermal cycler: It is critical that the thermal cycler be accurately programmed for
the correct cycle sequence, temperatures and the time for each of the cycles.
3. Oil or Wax: For thermal cyclers that do not have a top heating plate, the reaction in
the tubes must be overlaid with oil or wax to prevent evaporation.
4. Manual Water Bath PCR: Three water baths can be used as an alternative to a ther-
mal cycler for PCR, but results are more variable. Samples require oil or wax layers.
This method requires extra care and patience.
GEL PREPARATION AND STAINING
5. Concentrated agarose: Gels of higher concentration (> 0.8%) require special atten-
tion when dissolving or re-melting. Make sure that the solution is completely clear of
clumps or glassy granules. Distorted electrophoresis DNA band patterns will result
if the gel is not properly prepared.
6. Electrophoretic separation: The tracking dye should travel at least 4 cm from the
wells for adequate separation before staining.
7. Staining: Staining of higher concentration gels (> 0.8%) require special care to
obtain clear, visible results. After staining (15 to 30 min.) with InstaStain Ethidium
Bromide, examine the results using a UV (300 nm) transilluminator. Repeat the stain-
ing as required.
8. DNA ladders or markers: After staining the agarose gel, the DNA 100 bp ladder, 200
bp ladder, or Standard DNA Markers should be visible. If bands are visible in the lad-
der or marker and control lanes, but bands in the sample lanes are faint or absent,
it is possible that DNA was not successfully extracted from the cells. If the ladder
or marker, control and DNA bands are all faint or absent, potential problems could
include improper gel preparation, absence of buffer in the gel, improper gel staining
or a dysfunctional electrophoresis unit or power source.
PCR Experimental Success Guidelines
A
27
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document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
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Experiment
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Appendix
B
PREPARATION OF THE PCR REACTION:
1. The PCR reaction sample should be prepared as specied in the experiment in-
structions. Each PCR reaction sample contains three critical components:

PCR Reaction pellet
Primer mix
DNA for amplication
2. After adding the components of the PCR reaction sample, use clean forceps to
transfer one wax bead to the PCR tube. At the start of the PCR reaction, the wax
will melt and overlay the samples to prevent evaporation during heating.
POLYMERASE CHAIN REACTION CYCLING
3. In the three-waterbath PCR method, the PCR reaction sample is sequentially
cycled between three separate waterbaths, each set at different temperatures, for
a specied period of time. The sequential placement of the reaction sample in
the waterbaths maintained at three different temperatures constitutes one PCR
cycle. One example of a PCR cycle might be as follows:
94C for 1 minute
50C for 1 minute
72C for 1 minute
See experiment instructions for specic program requirements.
4. The PCR tube must be handled carefully when sequentially cycled between the
three waterbaths. For each cycle:
Carefully place the PCR tube in a waterbath oat. Make sure that the sample
volume is at the bottom of the tube and remains undisturbed. If necessary,
pulse spin the tube in a balanced microcentrifuge, or shake the tube to get all
of the sample to the bottom of the tube.
Use forceps to carefully lower the waterbath oat (with tubes) sequentially
into the waterbaths.
5. Process the PCR reaction sample for the total number of cycles specied in the ex-
periment instructions. On the nal cycle the 72C incubation can be extended
to 5 minutes.
6. After all the cycles are completed, the PCR sample is prepared for electrophoresis.
Polymerase Chain Reaction Using Three Waterbaths
Superior PCR results are obtained using an automated thermal cycler. However, if you do
not have a thermal cycler, this experiment can be adapted to use three waterbaths (Cat.
# 544). Much more care needs to be taken when using the three-waterbath PCR method.
The PCR incubation sample is small and can easily be evaporated. Results using three wa-
terbaths are often variable. Please refer to the Appendix entitled "PCR Samples with Wax
Overlays" for sample handling and preparation tips.
Each PCR Reaction pellet
contains Taq DNA poly-
merase, four deoxytriphos-
phates, Mg
+2
and buffer.
It is imperative that the
temperatures are accurately
maintained throughout the
experiment.
Important Note
28
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Experiment
Identication of Foodstuffs
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Appendix
C
Preparation and Handling of PCR Samples With Wax
For Thermal Cyclers without Heated Lids, or
PCR Using Three Waterbaths
Automated thermal cyclers with heated lids are designed to surround the entire sample tube at the appropri-
ate temperature during PCR cycles. Heating the top of the tubes during these cycles prevents the very small
sample volumes from evaporating. For thermal cyclers without heated lids, or when conducting PCR by the
three-waterbath method, it is necessary to add a wax bead to the reaction sample. During the PCR process, the
wax will melt and overlay the samples to prevent evaporation during heating.
Each PCR Reaction
pellet contains Taq
DNA polymerase, four
deoxytriphosphates,
Mg
+2
and buffer.
PREPARING THE PCR REACTION:
1. The PCR reaction sample should be prepared as specied in the experiment
instructions. Each PCR reaction sample contains the following three critical
components:

PCR Reaction pellet
Primer mix
DNA for amplication
2. After adding the components of the PCR reaction sample, use clean forceps to
transfer one wax bead to the PCR tube.
3. Process the PCR reaction sample for the total number of cycles specied in the
experiment instructions.
PREPARING THE PCR REACTION FOR ELECTROPHPORESIS:
4. After the cycles are completed, transfer the PCR tube to a rack and prepare
the PCR sample for electrophoresis.
Place the PCR tube in a 94C waterbath long enough to melt the wax over-
lay. Use a clean pipet to remove most of the melted wax overlay.
Allow a thin layer of the wax to solidify.
Use a clean pipet tip to gently poke a hole through the solidied wax.
Remove the tip.
Use another clean pipet tip to enter the hole to remove the volume of
mixture specied in the experiment instructions. Transfer this volume to a
clean tube.
Add other reagents according to experiment instructions, if applicable,.
Add 5 l of 10x Gel Loading solution to the sample and store on ice.
5. Proceed to delivery of the sample onto an agarose gel for electrophoresis as
specied in the experiment instructions.
29
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Experiment
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Appendix
Time and Voltage Guidelines
(2.0% Gel)
Time Volts
125
70
50
40 min
2.0 hrs
3.0 hrs
Approx. migration
distance of
tracking dye
Gel Size (cm)
Volume
7 x 7
25 ml
7 x 14
50 ml
125
70
50
60 min
2.5 hrs
3.5 hrs
4.5 cm
6.0 cm
Table
C.3
If preparing a 2.0% gel
with concentrated (50x)
buffer, use Table A.7.
If preparing a 2.0% gel
with diluted (1x) buffer, use
Table A.8.
2.0% Agarose Gel Electrophoresis Reference Tables
Time and Voltage recommendations for EDVOTEK
equipment are outlined in Table C.3 for 2.0%
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 recom-
mended electrophoresis buffer is
Tris-acetate-EDTA, pH 7.8. The
formula for diluting EDVOTEK
(50x) concentrated buffer is one
volume of buffer concentrate to
every 49 volumes of distilled or
deionized water. Prepare buffer
as required for your electropho-
resis unit.
D
Amt of
Agarose
(g)
Concentrated
Buffer (50x)
(ml)
Size of Gel
(cm)
Distilled
Water
(ml)
Total
Volume
(ml)
7 x 7
7 x 14
0.5
1.0
0.5
1.0
24.5
49.0
+
= +
Table
A.7
25
50
Individual 2.0%
UltraSpec-Agarose Gel
Table
A.8
Amt of
Agarose
(g)
Diluted
Buffer (1x)
(ml)
Size of Gel
(cm)
7 x 7
7 x 14
0.5
1.0
25
50
+
Individual 2.0%
UltraSpec-Agarose Gel
50x Conc.
Buffer (ml)
Distilled
Water (ml)
+
EDVOTEK
Model #
Total Volume
Required (ml)
Electrophoresis (Chamber) Buffer
M6+
M12
M36
Dilution
Table
B
300
400
1000
294
392
980
6
8
20
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Experiment
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Appendix
E
To save time, electrophoresis buffer and agarose gel solution can be prepared in larger
quantities for sharing by the class. Unused diluted buffer can be stored for use at a later
time and solidied agarose can be remelted.
Buffer and Agarose Quantity Preparations
BULK ELECTROPHORESIS BUFFER
Quantity (bulk) preparation for 3 liters of 1x electropho-
resis buffer is outlined in Table D.
Concentrated
Buffer (50x)
(ml)
Distilled
Water
(ml)
Total
Volume
(ml)
60 2,940 3000 (3 L)
=
+
Bulk Preparation of
Electrophoresis Buffer
Table
D
60C
8.0 8.0 392 400

Batch Preparation of
2.0% UltraSpec-Agarose
Amt of
Agarose
(g)
Concentrated
Buffer (50X)
(ml)
+
Distilled
Water
(ml)
Total
Volume
(ml)
= +
Table
E.3
PREPARING AGAROSE GELS BY BATCH
For quantity (batch) preparation of agarose gel solu-
tion, refer to Table E.3.
1. Use a 500 ml Pyrex ask or beaker to prepare the
diluted gel buffer
2. Pour the appropriate amount 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 in the same manner as described 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 dissipation 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.
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.
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.
31
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Experiment
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Appendix
F
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
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.
Agarose Gel Preparation
Step by Step Guidelines
Place one of
the black end
caps with the
wide u shaped slot facing 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.
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.
At high altitudes, it is recommended
to use a microwave oven to reach
boiling temperatures.
32
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Appendix
F
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
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.
33
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 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
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. If staining a 7 x 14 cm gel, use two 7 x 7 cm In-
staStain EtBr cards. If staining a 7 x 7 cm gel,
use one card.

Wearing gloves, remove the clear plastic protec-
tive sheet from the InstaStain EtBr card(s).
Place the unprinted side of the InstaStain EtBr
card(s) on the gel.
Visit our web site for an animated
demonstration of InstaStain EtBr.
Disposal of InstaStain
Disposal of InstaStain cards and gels should follow institutional guidelines for
chemical waste.
Caution: Ethidium Bromide is a listed mutagen.
Staining and Visualization of DNA
InstaStain Ethidium Bromide Cards
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.
Markers (Standard DNA Fragments, DNA 100 bp ladder or DNA 200 bp ladder) should be visible after stain-
ing even if the DNA samples are faint or absent. If markers are not visible, troubleshoot for problems with
the electrophoretic separation.
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 10-15 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 pro-
tective goggles.
Wear Gloves
and UV Safety
Goggles
G
DNA InstaStain
Patents Pending DNA InstaStain
Patents Pending
DNA InstaStain
Patents Pending
DNA InstaStain
Patents Pending
-
-
-
-
-
DNA InstaStain
Patents Pending DNA InstaStain
Patents Pending
-
-
-
-
-
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

Do not stain gel(s) in the
electrophoresis apparatus.
34
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Appendix
H
Staining and Visualization of DNA
FlashBlue Liquid Stain
Do not stain gel(s) in the
electrophoresis apparatus.
Staining and Destaining
1. Remove the agarose gel from its bed and completely submerse the
gel in a small, clean tray containing 75 ml of 1x FlashBlue stain.
Add additional stain if needed to completely submerge the gel.
2. Stain the gel for no more than 5 minutes.
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.
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.
7. If the gel is too light and bands are difcult to see, repeat the staining and
destaining procedures.
Storage and Disposal of 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.
35
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 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Appendix
Staining and Visualization of DNA
InstaStain Blue
One-step Staining and destaining
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.
Do not stain gel(s) in the
electrophoresis apparatus.
InstaStain
One Step
Stain and
Destain
1. Remove the 7 x 7 cm agarose gel from its bed and completely submerse the gel in
a small, clean tray containing 75 ml of distilled or deionized water, or used electro-
phoresis buffer. The agarose gel should be completely covered with liquid.
Examples of small trays include large weigh boats, or small plastic food containers
2. 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.
I
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.
36
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Appendix
TO MAXIMIZE SUCCESS:
1. The approximate time for electrophoresis will
vary from experiment to experiment. A variety
of factors, including gel concentration, will inu-
ence electrophoresis time. Generally, the higher
the voltage applied, the faster the samples mi-
grate. However, depending upon the apparatus
conguration and the distance between the two
electrodes, individual electrophoresis units will
separate DNA at different rates.
2. Do not move the apparatus after the samples
have been loaded.
Moving the apparatus will dislodge the
samples from the wells into the buffer and
will compromise results.
If it is absolutely necessary to move the
apparatus during electrophoresis, you may
safely do so after the tracking dye has mi-
grated at least 1 cm from the wells into the
gel.
3. For optimal DNA fragment separation, do not
use voltages higher than 125 volts for agarose
gel electrophoresis. Higher voltages can over-
heat and melt the gel.
4. The DNA samples contain tracking dye, which
moves through the gel ahead of most DNA (ex-
cept extremely small fragments). Migration of
the tracking dye will become clearly visible in the
gel after approximately 10-15 minutes.
5. If DNA fragments are similar in size, fragments
will migrate close to one another.
In general, longer electrophoretic runs will
increase the separation between fragments
of similar size.
Experiments which involve measurement of
fragment molecular size or weight should
be run at the recommended optimal time to
ensure adequate separation.
Figure is not
drawn to scale.
( + )
( - )
TRACKING
DYE
Migration
3.5 - 4.0 cm
Sample
wells
Agarose Gel Electrophoresis Hints and Help
J
M
o
n
- Fri 9 a
m

-

6

p
m

E
T
(1-800-338-6835)
E
D
V
O
-
T
E
C
H SERVIC
E
1-800-EDVOTEK
Mon - Fri
9:00 am to 6:00 pm ET
FAX: (301) 340-0582
Web: www.edvotek.com
email: info@edvotek.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
37
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 2004, 2005, 2006, 2007, 2008, 2010, 2011 EDVOTEK, Inc., all rights reserved. EVT 2011_08_22AM
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
962
Experiment
Identication of Foodstuffs
from Genetically Modied Organisms
Appendix
6. Electrophoresis should be terminated when the
tracking dye has moved a minimum of 3.5 to 4
centimeters from the wells for 7 x 7 cm gels, or
5-8 centimeters for 7 x 14 cm gels. Terminate the
electrophoresis before the tracking dye moves
off the end of the gel.
For optimal results, stain the gel immediately
after electrophoresis.
For convenience, the power source can be
connected to a household automatic light
timer to terminate the electrophoretic sepa-
ration and avoid running samples off the
end of the gel.
AVOIDING COMMON PROBLEMS
To avoid potential problems, some suggestions and
reminders are listed below.
7. Use only distilled or deionized water to prepare
buffers and gels. Do not use tap water.
8. To ensure that DNA bands are well resolved,
make sure the gel formulation is correct and that
electrophoresis is conducted for the recommend-
ed optimal amount of time.
9. Correctly dilute the concentrated buffer for
preparation of both the gel and electrophore-
sis (chamber) buffer. Remember that without
buffer in the gel, there will be no DNA mobil-
ity. Check that the gel is completely submerged
under buffer during electrophoresis.
10. For optimal results, use fresh electrophoresis buf-
fer prepared according to instructions.
11. Before performing the actual experiment, prac-
tice sample delivery techniques to avoid diluting
the sample with buffer during gel loading.
12. To avoid loss of DNA fragments into the buffer,
make sure the gel is properly oriented so the
samples are electrophoresed from the negative
electrode (cathode) towards the positive elec-
trode (anode).
13. To avoid obtaining gel results that are missing
small DNA fragments (small fragments move
faster), remember that the tracking dye in the
sample moves through the gel ahead of the
smallest DNA fragments. Terminate the electro-
phoresis before the tracking dye moves off the
end of the gel.
14. If DNA bands appear faint after staining and
destaining, repeat the procedure. Staining for a
longer period of time will not harm the gel. Re-
stained gels may require longer destaining.
CARE AND MAINTENANCE OF THE
ELECTROPHORESIS APPARATUS
15. The temperature of the melted agarose which
is poured into the bed during gel casting should
not exceed 60C. Hot agarose solution may ir-
reversibly warp the casting tray.
16. Avoid touching the fragile platinum electrodes.
17. Power should always be turned off and leads
disconnected from the power source when the
cover is removed from the apparatus.
18. To clean the apparatus chamber, gel casting tray
and combs, rinse thoroughly with tap water.
Give the items a nal rinse with distilled water.
Let them air dry. Do not use detergents of any
kind, or expose the apparatus to alcohols.
19. EDVOTEK injection-molded electrophoresis units
do not have glued junctions that can develop
potential leaks. In the unlikely event that a leak
develops in any electrophoresis apparatus you
are using, IMMEDIATELY SHUT OFF POWER. Do
not use the apparatus.
J
Agarose Gel Electrophoresis Hints and Help
continued
Material Safety Data Sheets
Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
962
Experiment
38
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39
962
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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o
t
e
: B
la
n
k
s
p
a
c
e
s
a
r
e
n
o
t
p
e
r
m
it
t
e
d
. If
a
n
y
it
e
m
is
n
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t

a
p
p
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a
b
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r
n
o
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t
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a
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o
p
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s
s


(
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t
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y
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m
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n
t
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t
y
;


C
o
m
m
o
n

N
a
m
e
(
s
)
]










O
S
H
A

P
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L
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T
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R
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O
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H
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2
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1
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t
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a
b
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a
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L
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s
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N
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la
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k
s
p
a
c
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s
a
r
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t
p
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m
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t
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m
is
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a
p
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a
b
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r
n
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f
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a
t
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a
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b
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m
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b
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t
.
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c
t
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n

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n
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s

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I
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H
a
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s

I
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f
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D

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a
z
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C
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m
p
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S
p
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c
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c

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h
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m
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c
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l

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d
e
n
t
i
t
y
;


C
o
m
m
o
n

N
a
m
e
(
s
)
]










O
S
H
A

P
E
L
A
C
G
I
H

T
L
V
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t
h
e
r

L
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m
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s

R
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c
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m
m
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d
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%

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A
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R
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(
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E
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M
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O
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c
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C
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d
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t
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t

O
c
c
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Material Safety Data Sheets
Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
962
Experiment
40
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C
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7
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C
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#
1
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