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EDVO-Kit #
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In Search of
the Cancer Gene
Storage: See Page 3 for
specific storage instructions
EXPERIMENT OBJECTIVE:
In this experiment, students will gain an understanding
of the effect of mutations in the p53 tumor suppressor
gene and its role in familial cancers.
This experiment is designed for DNA staining with InstaStain® Ethidium Bromide.
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Table of Contents
Page
Experiment Components
Experiment Requirements
Background Information
Experiment Procedures
Experiment Overview and General Instructions
Module One: Construction of a Family Pedigree
Module Two: Separation of DNA Fragments
Module Three: Analysis of Autorads to Search for
p53 Mutagens
Study Questions
Instructor's Guidelines
Notes to the Instructor
Experiment Results and Analysis
Study Questions and Answers
3
4
5
12
13
14
17
18
19
23
25
Appendices
A
Agarose Gel Preparation For DNA Staining with
InstaStain® Ethidium Bromide
28
B
Quantity Preparations for Agarose Gel Electrophoresis
29
C
Staining and Visualization of DNA with
InstaStain® Ethidium Bromide Cards
30
Material Safety Data Sheets
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In Search of the Cancer Gene
EDVO-Kit #
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Components & Requirements
Experiment Components
This experiment contains reagents
for 6 groups.
This experiment is designed for
DNA staining with InstaStain®
Ethidium Bromide.
Ready-to-Load Samples for Electrophoresis
A
B
C
D
E
Storage:
All DNA samples
can be stored in
the refrigerator
Standard DNA Fragments
Control DNA
Patient Peripheral blood DNA
Patient Breast Tumor DNA
Patient Normal Breast Tissue DNA
Reagents & Supplies
•
•
•
•
X-ray simulated p53 hot spot sequences
UltraSpec-Agarose™ powder
Concentrated electrophoresis buffer
InstaStain® Ethidium Bromide
Before use, check all volumes of Standard DNA
fragments and DNA samples for electrophoresis.
Evaporation may have caused samples to become
more concentrated.
Approximate
Volume
Measurements
0.5 cm tube
120 µl
1.0 cm
4.1 cm
All components are
intended for educational
research only. They are not
to be used for diagnostic or
drug purposes, nor administered to or consumed by
humans or animals.
If needed, tap
tubes or centrifuge,
then add distilled
water to slightly
above the 1.0 cm
level and mix.
THIS EXPERIMENT DOES
NOT CONTAIN HUMAN
DNA. None of the experi-
ment components are derived from human sources.
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Requirements
•
•
•
•
•
•
•
•
•
•
•
E
O
DV
-TE
C H S E RV I C E
Horizontal gel electrophoresis apparatus
D.C. power supply
Automatic micropipets with tips
Balance
Microwave, hot plate or burner
Pipet pump
250 ml flasks or beakers
Hot gloves
Safety goggles and disposable laboratory gloves
Distilled or deionized water
UV Transilluminator
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information ready:
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• Literature version number
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314
About Family Pedigrees
When drawing a family pedigree, the following are general guidelines to
the symbols used and their representations:
A Circle represents a female.
•
A square represents a male.
•
A shaded circle or square refers to a person having some form of cancer.
•
An open (non-shaded) square or circle
represents a person who is free of cancer.
•
A circle or square (either shaded or open)
with a diagonal slash through it represents a person who is deceased.
Male free of cancer
Female with some form of cancer
Male with some form of cancer
or
Female , deceased
or
Male, deceased
In Li-Fraumeni syndrome, the pattern of
cancers in family pedigrees suggest dominant
inheritance. It is a genetic predisposition
leading to specific types of cancers. Typically,
the onset of cancer is at an early age, with
multiple primary tumors.
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Background Information
Female free of cancer
•
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In Search of the Cancer Gene
EDVO-Kit #
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The Role of Genes in Cancer
Background Information
Many contributory factors have been identified to cause the onset of cancer
that include exposure to certain carcinogens in our diets and environment.
Several forms of cancer have familial predisposition. These cancers appear to
be linked to inherited mutation of suppressor genes, such as p53.
Hereditary
Sporadic
Germline Mutation
Somatic Mutation
Normal Gene
Somatic
Mutation
MultipleTumors
Bilateral Tumors
Early onset
Fi rs t Somatic
Mutation
Second Somatic
Mutation
Familial cancers constitute a
very small fraction of the total
reported cancers and occur in
dominant inherited patterns.
Mutations that are directly inherited are referred to as germline
mutations. Such mutations can
be detected in familial pedigrees.
A second type of mutation,
known as somatic mutations, do
not have direct genetic links and
are acquired during the life of
the individual. Patterns of typical
hereditary and sporadically acquired nonhereditary pedigrees
appear in Figure 1.
In a germline with an inherited
mutation, a single somatic mutation within a suppressor gene
will result in the inactivation of
both alleles. By contrast, normal inherited suppressor genes,
that are free of mutations, will
require two sequential mutations
to initiate tumors. This model
is referred to as the "Two-hit"
hypothesis.
Historically, some of the first
genes identified include the retinoblastoma (RB) gene, Wilm's’
tumor (WTI), neurofibromatosis
type II gene and Li-Fraumeni syndrome. In Li-Fraumeni syndrome,
Figure 1: Hereditary and Sporadic Models of Gene Inactivation.
a notable feature in family pedigrees, include a sarcoma patient
and at least two immediate relatives with other cancers before the age of 45, as well as multiple cancers in
other family members. This is illustrated in figure 2.
Single Tumors
Unilateral Tumors
Later onset
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the written consent of EDVOTEK, Inc. Copyright © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
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314
The Role of Genes in Cancer
The Human Genome Project has provided
information to link identification of various
cancers and other diseases to DNA sequencing information. This information needs to
be handled cautiously to assure confidentiality of patient genetic profiles.
BB.42
BR.36
SS.23
BB.34
CN.2
CN.36
OS.13
BB
BR
CN
LK
OS
SS
LK.2
Bilateral breast cancer
Breast cancer
Brain tumor
Leukemia
Osteosar coma
Soft tissue sar coma
The study of inherited cancers has given
cancer molecular biologists the opportunity
to search for genes that are critical in normal
cell development and carcinogenesis. At the
molecular level, cancer formation is characterized by alterations in both dominant
oncogenes and tumor suppressor genes,
such as p53. Suppressors are normal cellular
proteins that are involved in limiting cell
growth. By contrast, oncogenes are involved
in promoting the growth of cells.
Figure 2: Example of a Li-Fraumeni family pedigree.
In recent years, the p53 tumor suppressor protein has become the center
of many cancer biology studies. Because it appears to be of major significance, there is great impetus to study how this gene functions in normal cells
compared to cancer cells. The gene for the p53 protein is located on the
short arm of chromosome 17. It encodes a normal 53,000 molecular nuclear
phosphoprotein. Wild type p53 functions as a cell regulator. There is now
well-documented evidence that normal p53 is a sequence-specific DNA-binding protein that is a transcriptional regulator. Upon introduction of mutations, p53 loses its ability to bind to DNA. By contrast, p53 that have mutations in specific hot spots promote uncontrolled cell growth and therefore
function as oncogenes. For a tumor suppressor gene such as p53 to play a
role in transformation in cancer, both alleles need to be altered, as shown in
figures 1 and 2.
The p53 protein can be divided into three domains. The first is the amino
terminus region which contains the transcriptional activation region. The
second is the central region within the protein where the majority of critical
"hot spot" mutations are located. These "hot spots" are sites where muta-
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Background Information
With the advent of molecular biology applications to medicine, gene maps
and the chromosomal locations of genes are available as tools for the
identification of predisposition for various diseases. The procedures used
to obtain such information include DNA isolation and the analysis of point
mutations in hot spot areas in cancer-related genes, such as p53. Several
methods of analysis for the detection of point mutations in genes include
DNA sequencing.
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In Search of the Cancer Gene
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Background Information
The Role of Genes in Cancer
tions are detected in high frequencies. They are between exons 5 through 8
where 95% of the mutations occur. Within this region there are five subregions where point mutations are detected in human cancers. The third
region of the p53 protein is the carboxyl section, the most complex section
that contains the oligomerization and nuclear localization sequences.
Examples of hot spots include codons 165 and 175 in exon 5; 196 and 213 in
exon 6; 245 and 248 in exon 7; 273 and 282 in exon 8; all are within the p53
protein. Several of these mutations result in an altered p53 protein conformation. In turn, these changes can result in increased stability of the mutant
protein and the ability to bind to the normal p53 protein and inactivate it.
It is of interest to note that there are correlations between mutation and
tumor tissue. One example is the mutation at amino acid 175 which is common in colon carcinoma but is rarely observed in lung carcinoma.
The inherited Li-Fraumeni syndrome as it has become to be known is rare.
When it does occur it affects young family members and results in high mortality rates. Two physicians, Li and Fraumeni first described the syndrome
after examining death certificates of 648 childhood sarcomas. It was discovered in four families where siblings and cousins had childhood sarcomas.
Further analysis showed more than 50% of the affected families had extended phenotypes that included brain, breast cancers and leukemias. Cells from
individuals with LFS have only a single wild type p53 allele. Examination of
their p53 gene have shown correlations of the cancers to mutations in the
protein as described above.
OVERVIEW OF POLYMERASE CHAIN REACTION:
PCR has two important advantages. The first is sensitivity, which allows for
DNA fingerprinting identification using much smaller amounts of DNA since
PCR amplifies DNA. The second advantage is the speed of PCR analysis,
which allows critical questions to be answered more quickly as compared to
Southern Blot analysis.
PCR amplification requires the use of a thermostable DNA polymerase, such
as Taq polymerase. Purified from a bacterium known as Thermus Aquaticus
that inhabits hot springs, Taq polymerase is commonly used in PCR because
it remains stable at near-boiling temperatures. Also included 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 amplified,
are incubated in an appropriate buffer that contains Mg2+. The primers are
designed to correspond to the start and end of the DNA to be amplified,
known as the "target".
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314
The Role of Genes in Cancer
= Separation of
2 DNA strands
= Primer 1
= Primer 2
Target Sequence
3'
5'
5'
3'
The Experiment
5'
3'
Denature 94°C
Cycle 1
3'
5'
5'
5'
3'
3'
Cycle 3
Cycle 2
3'
3'
3'
5'
5'
5'
5'
5'
5'
5'
5'
3'
5'
5'
5'
3'
5'
3'
5'
3'
5'
5'
5'
3'
5'
5'
5'
3'
5'
5'
5'
5'
5'
Anneal
2 primers
45°C
5'
3'
5'
5'
3'
5'
3'
5'
3'
5'
3'
Extension
72°C
3'
3'
Figure 3: The Polymerase Chain Reaction
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The Role of Genes in Cancer
The Experiment
The PCR reaction mixture (which contains the DNA polymerase, buffer,
deoxynucleotides, primers, and template) is subjected to sequential heating/
cooling cycles at three different temperatures (Figure 3).
•
In the first step, the enzyme reaction is heated to near boiling (92°
- 96°C.) to denature or "melt" the DNA. This step, known as "denaturation" disrupts the hydrogen bonds between the two complimentary
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 to 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 72°C. At this temperature the Taq
polymerase is maximally active and adds nucleotides to the primers to
synthesize the new complimentary strands.
OVERVIEW OF DNA SEQUENCING:
During DNA sequence analysis, four separate enzymatic reactions are performed, one for each nucleotide. Each reaction contains DNA polymerase,
the single-stranded DNA template to be sequenced and to which the
synthetic DNA primer has been hybridized, the four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, dTTP), 32P labeled or fluorescent nucleotide(s)
are added for the detection of growing DNA fragments (Figure 4), and the
appropriate buffer for in vitro DNA synthesis.
In sequencing reactions, the “G” reaction contains dideoxyGTP, the “C” reaction dideoxyCTP, the “A” reaction dideoxyATP, and the “T” reaction dideoxyTTP. The dideoxynucleotide concentrations are carefully adjusted so that
they are incorporated into a growing DNA strand randomly and infrequently. Once a dideoxynucleotide is incorporated into the growing DNA strand,
DNA synthesis is terminated. The site of the dideoxynucleotide incorporation allows one to determine the position of that base. The dideoxynucleotide lacks a 3'-OH group on the ribose ring and as a consequence, DNA
synthesis is terminated because the DNA polymerase will not add another
nucleotide to the growing strand since a 3'-OH group is absolutely required
for DNA chain elongation.
Since a particular reaction will contain millions of growing DNA strands, a
“nested set” of fragments is obtained with each fragment being terminated
at a different position corresponding to the random incorporation of the
dideoxynucleotide.
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The Role of Genes in Cancer
C
T
A
G
Deduced
Sequence
3’
5’
G
A
G
C
C
A
G
C
G
A
G
T
A
Fragment
Size
31
30
29
28
27
26
25
24
23
22
21
20
19
It should be apparent
that together the “G, A,
T, C” “nested sets” contain fragments ranging in
size successively from 19
to 31 nucleotides for the
hypothetical sequence
in Figure 3. In the figure
the “G” reaction contains
fragments of 21, 23, 25,
29 and 31 nucleotides in
length. The first eighteen
nucleotides are contained in
the synthetic DNA sequencing primer which are not
shown. The rest are added
during de novo synthesis by
the DNA polymerase.
The reaction products from
the G, A, T, and C reactions
are separated by electroFigure 4: Determining the sequence of a DNA fragment.
phoresis using a thin and
long vertical polyacrylamide
gel. DNA sequencing gels
resolve fragments which differ in size by a single nucleotide. After electrophoretic separation is complete, the sequence is determined by either
radioactivity or fluorescence. If radioactivity was used, autoradiography is
performed. The polyacrylamide gel is placed into direct contact with a sheet
of x-ray film. Since the DNA fragments are radioactively labeled with 32P,
their position can be detected by a dark exposure band on the sheet of x-ray
film. Recent advances have also made it possible to automate DNA sequencing and to avoid the use of isotopic 32P.
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The Experiment
Figure 4 shows a “nested set” of fragments produced for a hypothetical
DNA sequence. The "G" reaction contains the standard reaction mixture
(dATP, dCTP, dGTP, dTTP, the DNA polymerase, the appropriate buffer for
DNA synthesis, 32P labeled or fluorescent tagged nucleotide(s) and a small
amount of dideoxyGTP (ddGTP). The ddGTP (dideoxyGTP) random and
infrequent incorporation will produce a “nested set” of fragments which
terminate with a ddGTP. The “nested set” is complimentary to the sequence.
Similar “nested sets” are produced in the separate “A”, “T”, and “C” reactions. For example, the “A” “nested set” would terminate with a ddATP.
12
In Search of the Cancer Gene
EDVO-Kit #
314
Experiment Overview and General Instructions
The Experiment
EXPERIMENT OBJECTIVE:
In this experiment, you will construct a family pedigree that is suspected to
have the classic Li-Fraumeni syndrome. DNA samples which have been enzymatically digested will be separated by electrophoresis on an agarose gel as
an independent diagnostic test. DNA sequencing x-rays simulating p53 hot
spot sequences will also be examined and mutations will be identified.
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 reflects 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 reflect this change.
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In Search of the Cancer Gene
EDVO-Kit #
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314
Module One: Construction of a Family Pedigree
A first step in the search and assignment of Li-Fraumeni syndrome is to establish the family pedigree of the patient.
The first part of the experiment is based on the information made available as part of a diagnosis by the family physician and the oncologist. The
pedigree information that you will develop is for a young woman who is
suspected to have the Li-Fraumeni syndrome.
Upon monthly breast self-examination, Valerie Brown, age 36, found a small
irregular mass. She was concerned because she knew that her mother had a
mastectomy when she was in her late thirties. Valerie made an appointment
with her physician, who referred her to a specialist at a local cancer center,
where she was diagnosed as having breast cancer. As part of the medical
work-up, the oncologist had inquired about her family history of cancer.
Upon consultation with her mother, Valerie learned that her father and his
family appeared to be free of cancer. However, in Valerie's mother's family,
several cases of cancer have occurred.
With the information given below, chart the family pedigree.
•
Her mother, Diane, was diagnosed and treated for breast cancer at the
age of 39.
•
Valerie did not know that Diane had a sister, Mabel, who died at age 2
of a brain tumor.
•
Diane's brother, James (age 40), underwent surgery, followed by chemotherapy for colon cancer.
•
Her maternal grandmother, Elsie, died at age 42 from bilateral breast
cancer.
•
Her maternal grandfather, Elmer, was free of cancer and is 88 years old.
•
Her maternal cousin, Patrick (son of James), died of brain cancer at 14.
•
Her cousin (Patrick's sister), Jane, was diagnosed with childhood leukemia and subsequently died at age 2 .
•
Patrick's two other brothers, Richard (age 28) and Curtis (age 30), are in
good health and free of cancer.
•
Valerie's sister, Nancy (age 38), is free of cancer.
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The Experiment
INFORMATION FOR DEVELOPING THE FAMILY PEDIGREE:
14
In Search of the Cancer Gene
EDVO-Kit #
314
The Experiment
Module One: Construction of a Family Pedigree
•
Nancy's son, Michael, was diagnosed at age 3 as having sarcoma. Recently, at age 18, he was diagnosed as having osteosarcoma.
•
Nancy's other son, John (age 16), and daughter, Jessica (age 8), are free
of cancer.
Valerie has five children:
•
•
•
•
•
Justin (age 16)
Sheila (age 14)
Robert (age 10)
Angela (age 8)
Anthony (age 6).
All the children show no signs of cancer at this time. Valerie has requested
that DNA sequencing be done for each of her children (see Module Three).
Module Two: Separation of DNA Fragments
The familial pedigree in Module 1 strongly suggests Li-Fraumeni syndrome.
In such a case, a secondary diagnostic test is normally conducted. In this
scenario, Valerie provides a sample of blood and tumor biopsy tissue to conduct DNA analysis for the p53 gene. Normally the procedure is to amplify
the gene using polymerase chain reaction. This is followed by one of several
methods to detect the presence of a point mutation at the hot spots.
In this simulation experiment, Valerie's DNA has already been digested with
a restriction enzyme that recognizes the mutant sequence at the simulated
hot spot site at nucleotide 165 which is also the palindrome CAGCTG for
the restriction enzyme. This restriction enzyme was used as a probe to cut
the simulated amplified gene for Valerie’s DNA sample, together with a
normal control and a set of standard DNA marker fragments. Digestion of
the normal amplified DNA will give a characteristic "control" DNA fragment
banding pattern. The DNA obtained from blood lymphocytes will give an
altered band pattern representing one normal allele and the second which
is the mutant. The DNA analysis from the tumor tissue will show only the
pattern for the tumor allele. The predigested DNA samples with the control
wild type and DNA markers will be separated by agarose gel electrophoresis,
stained, and then analyzed.
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314
Module Two: Separation of DNA Fragments
Important Note
Continue heating until the
final solution appears clear
(like water) without any undissolved particles. Check
the solution carefully. If you
see "crystal" particles, the
agarose is not completely
dissolved.
AGAROSE GEL REQUIREMENTS FOR THIS EXPERIMENT
•
Recommended gel size:
7 x 7 cm
•
Number of sample wells required:
5
•
Agarose gel concentration:
0.8%
PREPARING THE AGAROSE GEL
1.
Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams or tape.
2.
Place a well-former template (comb) in the first set of notches at the end
of the bed. Make sure the comb sits firmly and evenly across the bed.
3.
To a 250 ml flask or beaker, add agarose powder and buffer as indicated
in the Reference Tables (Appendix A) provided by your instructor. Swirl
the mixture to disperse clumps of agarose powder.
4.
With a marking pen, indicate the level of the solution volume on the
outside of the flask.
5.
Heat the mixture using a microwave oven or burner to dissolve the agarose powder.
6.
Cool the agarose solution to 60°C 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 4.
After the gel is cooled to 60°C:
7.
Place the bed on a level surface and pour the cooled agarose solution
into the bed.
8.
Allow the gel to completely solidify. It will become firm and cool to the
touch after approximately 20 minutes.
9.
After the gel is solidified, be careful not to damage or tear the wells
while removing the rubber dams or tape and comb(s) from the gel bed.
10. Place the gel (on its bed) into the electrophoresis chamber, properly
oriented, centered and level on the platform.
11. Fill the electrophoresis apparatus chamber with the appropriate amount
of diluted (1x) electrophoresis buffer (refer to Table B on the instruction
sheet from the Appendix provided by your instructor).
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
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The Experiment
If you are unfamiliar
with agarose gel
preparation and
electrophoresis,
detailed instructions
and helpful resources
are available at
www.edvotek.com
16
In Search of the Cancer Gene
EDVO-Kit #
314
Module Two: Separation of DNA Fragments
LOADING THE SAMPLES
The Experiment
This experiment is designed for staining with InstaStain® Ethidium Bromide.
The amount of sample that should be loaded is 18-20 µl. Make sure the gel
is completely submerged under buffer before loading the samples and conducting electrophoresis.
Reminder:
LOADING DNA SAMPLES
12. Heat the DNA fragments (tubes A-E) for
two minutes at 65°C. Allow samples to
cool for a few minutes before gel loading.
13. Load 18-20 µl of each DNA sample in the
following manner:
Lane
Tube
1
2
3
4
5
A
B
C
D
E
Before loading the samples,
make sure the gel is
properly oriented in the
apparatus chamber.
–
Black
Sample wells
+
Red
Standard DNA Fragments
Control DNA
Patient Peripheral Blood DNA
Patient Tumor DNA
Patient Breast Normal DNA
RUNNING THE GEL
14. After the DNA samples are loaded, properly orient the cover and carefully snap it onto the electrode terminals.
Electrophoresis can
be completed in
15-20 minutes under
optimal conditions.
For Time and Voltage
recommendations, refer
to Table C (Appendix A).
15. Insert the plugs of the black and red wires into the corresponding inputs
of the power source.
16. Set the power source at the required voltage and conduct electrophoresis for the length of time determined by your instructor.
17. Check to see that current is flowing properly - you should see bubbles
forming on the two platinum electrodes.
18. After the electrophoresis is completed, disconnect the power and remove the gel from the bed for staining.
STAINING AND VISUALIZATION OF DNA
After electrophoresis, agarose gels require staining to visualize the separated
DNA samples. Your instructor will provide instructions for DNA staining with
InstaStain® Ethidium Bromide (Appendix C).
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
reserved
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In Search of the Cancer Gene
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314
Module Three: Analysis of Autorads to Search for p53 Mutagens
In this part of the experiment, x-ray results of the wild p53 and samples
from Valerie's five children will be read to determine whether or not there
are mutations.
Ages
Justin
Sheila
Robert
Angela
Anthony
16
14
10
8
6
Autorad #
1
2
3
4
5
1.
For each of Valerie's children, obtain the appropriate sample autoradiograph and place it on a light box to enhance visualization.
2.
The sequencing reactions have all been loaded in order: G-A-T-C.
3.
Begin analysis of the DNA sequence at the bottom of the autoradiograph with the circled band, which is an A.
4.
Compare the deduced sequence to the wild type sequence shown in the
box below.
5.
Identify the location of the potential mutant nucleotide. What was the
mutation? Is there more than one mutation?
Wild Type sequence:
5'-AGCTTGGCTGCAGGTCGACGGATCCCCAGGAATTCGTAAT-3'
6.
Based on the information obtained from the x-rays, which of Valerie's
children have a mutation in their DNA sequence?
Note: This is a simulation and the DNA sequence is not that of
p53. The principles of reading DNA sequences and finding the point
mutation is the same.
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
reserved
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The Experiment
Valerie's children
18
In Search of the Cancer Gene
EDVO-Kit #
314
Study Questions
The Experiment
Answer the following study questions in your laboratory notebook or on a
separate worksheet.
1.
What is the difference between tumor suppressors and oncogenes?
2.
What are the effects of hot spots in p53 protein structure?
3.
Why does Valerie's tumor DNA sample have fewer bands than the peripheral blood?
4.
What is the purpose of the control lane?
5.
Can a physician proceed with diagnosis based on molecular biology
data?
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
reserved
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In Search of the Cancer Gene
EDVO-Kit #
314
Instructor’s 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
fit your specific set of circumstances. If you do not find the answers to your
questions in this section, a variety of resources are continuously being added
to the EDVOTEK web site. In addition, Technical Service is available from
9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at 1-800-EDVOTEK (1-800-338-6835).
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 explanation. Please visit our website for specific content and skill standards for
various experiments.
EDUCATIONAL RESOURCES
Electrophoresis Hints, Help and Frequently Asked Questions
The EDVOTEK web site provides several suggestions and reminders for conducting electrophoresis, as well as answers to frequently asked electrophoresis questions.
E
O
DV
-TE
C H S E RV I C E
Technical Service
Department
Mon - Fri
9:00 am to 6:00 pm ET
Online Ordering
now available
1-800-EDVOTEK
ET
(1-800-338-6835)
Mo
FAX: (301) 340-0582
Web: www.edvotek.com
email: info@edvotek.com
pm
6
n - Fri 9 am Please have the following
Visit our web site for information
about EDVOTEK’s complete line
of “hands-on” experiments for
biotechnology and biology education.
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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In Search of the Cancer Gene
EDVO-Kit #
314
Notes to the Instructor:
HOW THIS EXPERIMENT IS ORGANIZED:
This experiment module has three modules:
1.
The Experiment
2.
3.
Determine the family pedigree of a family suspected of suffering with
LFS and identifying the cancer patients.
Separate DNA with amplified p53 genes predigested with a restriction
enzyme probe to detect a specific point mutation within the p53 gene.
Examine x-rays of DNA sequence information to identify the mutation
of a hot spot codon. This is a simulation of data for 5 children of the
patient.
A brief overview of DNA sequencing is included as a refresher in the Background Information section.
MICROPIPETTING BASICS AND PRACTICE GEL LOADING
Accurate pipeting is critical for maximizing successful experiment results.
EDVOTEK 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 recommends 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
•
Pre-lab preparations
Pre-lab preparations and dispensing of biologicals and reagents take approximately 1-2 hours.
•
Table
C
Agarose Gel preparation
Whether you choose to prepare the gel(s) in advance or have the
students prepare their own, allow approximately 30-40 minutes for
this procedure. Generally, 20 minutes of this time is
required for gel solidification. See section “Options for
Time and Voltage
Preparing Agarose Gels” below.
Recommendations
EDVOTEK Electrophoresis Model
Volts
M6+
M12 & M36
Minimum / Maximum
Minimum / Maximum
150
15 / 20 min
25 / 35 min
125
20 / 30 min
35 / 45 min
70
35 / 45 min
60 / 90 min
50
50 / 80 min
95 / 130 min
•
Conducting Electrophoresis
The approximate time for electrophoresis will vary from
15 minutes to 2 hours. Generally, the higher the voltage applied, the faster the samples migrate. However,
depending upon the apparatus configuration 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.
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
reserved
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In Search of the Cancer Gene
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314
Notes to the Instructor:
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
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. Solidified gels can
be stored under buffer in the refrigerator for up to 2 weeks.
Do not store gels at -20°C. Freezing will destroy the gels.
Gels that have been removed from their trays for storage, 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 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 0.8%. Prepare gels according to Table A.1 or A.2 in Appendix A.
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
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Instructor’s Guide
gels for the experiment.
22
In Search of the Cancer Gene
EDVO-Kit #
314
Notes to the Instructor:
Instructor’s Guide
GEL STAINING AND DESTAINING AFTER ELECTROPHORESIS
After electrophoresis, the agarose gels require staining in order to visualize the separated DNA samples. This experiment features staining with a
proprietary stain called InstaStain® Ethidium Bromide (InstaStain® EtBr).
Instructions for staining with InstaStain® are outlined in Appendix C. 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 staining procedures. Disposal of
InstaStain® cards and gels should follow institutional guidelines for chemical
waste.Caution: Ethidium Bromide is a listed mutagen.
PHOTODOCUMENTATION OF DNA (OPTIONAL)
There are many different photodocumentation systems available, including
digital systems that are interfaced directly with computers. Specific instructions will vary depending upon the type of photodocumentation system you
are using.
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
reserved
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In Search of the Cancer Gene
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314
Experiment Results and Analysis
MODULE ONE - PEDIGREE
Elsie BB, 42
James CC, 40
Instructor’s Guide
Elmer, 88
Diane BR, 39
Mabel CN, 2
Valerie
BR, 36
Jane Patrick Richard Curtis
LK, 2 CN, 14
38
30
Sheila
14
Justin
16
Robert Angela Anthony
6
10
8
Nancy
38
Michael
CS, 3
OS, 18
John
16
Jessica
8
MODULE TWO - AGAROSE GEL ELECTROPHORESIS
The schematic depicts an idealized gel result.
(-)
1 2 3
4 5 6
23130 bp
9416 bp
6557 bp
4361 bp
3000 bp
Lane
1
2
3
4
5
Tube
A
B
C
D
E
Standard DNA Size Fragments
Control Normal DNA
Patient (Valerie's) Peripheral Blood DNA
Patient (Valerie's) Breast Tumor DNA
Patient (Valerie's) Normal Breast Normal
2322 bp
2027 bp
725 bp
570 bp
(+)
Actual results will yield broader bands of varying intensities. Smaller
fragments will stain less efficiently and will appear as fainter bands.
The idealized schematic shows the relative positions of the bands, but
are not depicted to scale.
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
reserved
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24
In Search of the Cancer Gene
EDVO-Kit #
314
Experiment Results and Analysis
EXPLANATION OF GEL RESULTS:
Instructor’s Guide
Lane 2: The normal (unmutated) p53 allele possesses no restriction sites for
the enzyme.
Lane 3: Valerie's normal tissue possesses one normal p53 allele and one
mutated p53 allele that is point-mutated to acquire a restriction site for the
enzyme. As shown in Figure 1 (the "two-hit" model, page 5), she inherits
one "hit" in all her somatic cells (including peripheral blood). as she still possesses a normal p53 allele in this tissue, tumor formation remains suppressed.
Lane 4: Valerie's tumor biopsy shows only the mutated p53 pattern, present now on both alleles. She now has acquired a second "hit" in this tissue,
which then becomes cancerous as she no longer possesses a normal p53
tumor suppressor.
Lane 5: Valerie's normal breast tissue DNA is the same as the peripheral
blood DNA, with one mutated and one normal p53 allele. This tissue is not
cancerous as one normal tumor suppressor is still present.
Results for Module Three
Sheila (#2), Angela (#4), and Anthony (#5) have a normal DNA sequence for
p53. Justin (#1) and Robert (#3) have a mutation in their DNA sequence,
thus increasing their chance for developing a cancer at some point during
their lifetimes.
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
reserved
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Please refer to the kit
insert for the Answers to
Study Questions
26
EDVO-Kit #
In Search of the Cancer Gene
314
xxx
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In Search of the Cancer Gene
EDVO-Kit #
314
Appendices
Appendices
A
Agarose Gel Preparation For DNA Staining with
InstaStain® Ethidium Bromide
B
Quantity Preparations for Agarose Gel Electrophoresis
C
Staining and Visualization of DNA with
InstaStain® Ethidium Bromide Cards
Material Safety Data Sheets
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27
28
Appendix A
EDVO-Kit #
314
0.8% Agarose Gel Preparation
DNA Staining with InstaStain® Ethidium Bromide
If preparing the gel with
diluted (1x) buffer, use
Table A.2.
If preparing the gel with
concentrated (50x) buffer,
use Table A.1.
Table
A.2
Table
A.1
Individual 0.8%*
UltraSpec-Agarose™ Gel
DNA Staining with
InstaStain® Ethidium Bromide
Individual 0.8%* UltraSpec-Agarose™ Gel
DNA Staining with InstaStain® EtBr
Size of Gel
(cm)
Amt of
Distilled
Concentrated
Total
Agarose + Buffer (50X) + Water = Volume
(g)
(ml)
(ml)
(ml)
7x7
0.2
0.5
24.5
25
7 x 14
0.4
1.0
49.0
50
Size of Gel
(cm)
Diluted
Amt of
Agarose + Buffer (1x)
(ml)
(g)
7x7
0.2
25
7 x 14
0.4
50
* 0.77 UltraSpec-Agarose™ gel percentage rounded up to 0.8%
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 concentrate to
every 49 volumes of distilled or
deionized water. Prepare buffer
as required for your electrophoresis unit.
Table
C
Table
B
EDVOTEK
Model #
300
6
294
M12
400
8
392
M36 (blue)
500
10
490
M36 (clear)
1000
20
980
Time and Voltage
Recommendations
M6+
Dilution
Total Volume
50x Conc.
Distilled
Required (ml) Buffer (ml) + Water (ml)
M6+
EDVOTEK Electrophoresis Model
Volts
Electrophoresis (Chamber) Buffer
M12 & M36
Minimum / Maximum
Minimum / Maximum
150
15 / 20 min
25 / 35 min
125
20 / 30 min
35 / 45 min
70
35 / 45 min
60 / 90 min
50
50 / 80 min
95 / 130 min
Time and Voltage recommendations for EDVOTEK
equipment are outlined in Table C. The approximate time for electrophoresis will vary from approximately 15 minutes to 2 hours depending upon
various factors. Conduct electrophoresis for the
length of time determined by your instructor.
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
reserved
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Appendix B
29
EDVO-Kit #
314
0.8% Agarose Gel Preparation
Quantity Preparations
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 used at a later time and solidified agarose gel can be remelted.
BULK ELECTROPHORESIS BUFFER
Table
D
Bulk Preparation of
Electrophoresis Buffer
Concentrated
Buffer (50x) +
(ml)
60
Distilled
Water
(ml)
2,940
=
Total
Volume
(ml)
3000 (3 L)
Table
Batch Preparation of
0.8%* UltraSpec-Agarose™
E
Amt of
Agarose
(g)
3.0
+
Distilled
Total
Concentrated
Buffer (50x) + Water = Volume
(ml)
(ml)
(ml)
7.5
382.5
Quantity (bulk) preparation for 3 liters of 1x electrophoresis buffer is outlined in Table D.
BATCH AGAROSE GELS (0.8%)
For quantity (batch) preparation of 0.8% agarose
gels, see Table E.
1.
Use a 500 ml flask to prepare the diluted gel buffer
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 flask.
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 60°C
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
flask in step 3.
390
*0.77% UltraSpec-Agarose™ gel percentage rounded up to 0.8%
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
specified or if the bottle's plastic seal has been broken,
weigh the agarose to ensure you are using the correct
amount.
60˚C
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 firm and cool to the touch after approximately 20 minutes. Then proceed with preparing
the gel for electrophoresis.
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
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30
Appendix C
EDVO-Kit #
314
Staining and Visualization of DNA
INSTASTAIN® ETHIDIUM BROMIDE CARDS
1.
2.
1
After electrophoresis, place the gel on a piece
of plastic wrap on a flat surface. Moisten the
gel with a few drops of electrophoresis buffer.
Wearing gloves, remove the clear plastic protective sheet, and place the unprinted side of
the InstaStain® EtBr card on the gel.
Moisten
the gel.
Wear gloves
and safety goggles
3.
Firmly run your fingers 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.
Do not stain gel(s) in the
electrophoresis apparatus.
2
™
Stain
nt
Pate
g
ndin
s Pe
Place the InstaStain®
card on the gel.
3
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.
Visit our web site for an animated
demonstration of InstaStain® EtBr.
a
Inst
DNA InstaStain
™
Patents Pending
Press firmly.
4
-
InstaStain™
Caution: Ethidium Bromide is a listed mutagen.
Disposal of InstaStain
Disposal of InstaStain® cards and gels should follow institutional guidelines for
chemical waste.
g
Patents Pendin
Place a small weight to
ensure good contact.
5
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
View on U.V. (300 nm)
transilluminator
the staining time an additional 10-15 minutes.
•
Gels stained alternatively with InstaStain Methylene Blue or liquid methylene blue may fade with time. Re-stain
the gel to visualize the DNA bands.
•
DNA 200 bp markers should be visible after staining even if the amplified DNA samples are faint or absent. If
markers are not visible, troubleshoot for problems with the electrophoretic separation.
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 © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights
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®
Agarose
Date Prepared
10/05/06
OSHA PEL
ACGIH TLV
% (Optional)
No data
Flammable Limits
LEL
UEL
No data available
Stable
Unstable
Will Not Occur
May Occur
Inhalation?
Ingestion?
Yes
No data available
OSHA PEL
ACGIH TLV
Unstable
Normal solid waste disposal
None
None
Yes
Other
Mechanical Gen. dilution ventilation
None
Skin: Wash with soap and water
None
Impervious clothing to prevent skin contact
Splash proof goggles
None
Yes
Work/Hygienic Practices
None
Other Protective Clothing or Equipment None
Protective Gloves
Mechanical (General)
Local Exhaust
Respiratory Protection (Specify Type)
Ventilation
Yes
Yes
Avoid eye and skin contact.
Section VIII - Control Measures
Other Precautions
Eye Protection
Other
Special
_Safety goggles
None
None
Dispose in accordance with all applicable federal, state, and local
enviromental regulations.
Precautions to be Taken in Handling and Storing
Waste Disposal Method
Wear suitable protective clothing. Mop up spill
and rinse with water, or collect in absorptive material and dispose of the absorptive material.
Date Prepared
OSHA PEL
Data not available
ACGIH TLV
CAS# 139-33-3
Soluble
No data
No data
No data
No data
X
N.D.
May Occur
Will Not Occur
Inhalation? Yes
X
Skin?
None
Conditions to Avoid
Yes
IARC Monographs?
No data
Treat symptomatically and supportively
Mix material with combustible solvent and burn in a chemical incinerator
equipped afterburner and scrubber
Rubber
Work/Hygienic Practices
Yes
SCBA
No
Eye Protection
Other
Special
Chem. safety goggles
None
Chem. fume hood
Use in chemical fume hood with proper protective lab gear.
Rubber boots
Mechanical (General)
Local Exhaust
Other Protective Clothing or Equipment
Protective Gloves
Ventilation
Respiratory Protection (Specify Type)
Section VIII - Control Measures
Mutagen
Use in chemical fume hood with proper protective lab gear.
Precautions to be Taken in Handling and Storing
Waste Disposal Method
Wear SCBA, rubber boots, rubber gloves
Steps to be Taken in case Material is Released for Spilled
Section VII - Precautions for Safe Handling and Use
Emergency First Aid Procedures
OSHA Regulation?
Irritation to mucous membranes and upper respiratory tract
Medical Conditions Generally Aggravated by Exposure
Signs and Symptoms of Exposure
Carcinogenicity: No data available NTP?
Other Precautions
N.D.
UEL
Ingestion? Yes
Health Hazards (Acute and Chronic) Chronic: May alter genetic material
Acute: Material irritating to mucous membranes, upper respiratory tract, eyes, skin
Route(s) of Entry:
Section VI - Health Hazard Data
Hazardous
Polymerization
Carbon monoxide, Carbon dioxide, nitrogen oxides, hydrogen bromide gas
Hazardous Decomposition or Byproducts
None
Conditions to Avoid
Strong oxidizing agents
Stable
Unstable
Section V - Reactivity Data
Emits toxic fumes
Unusual Fire and Explosion Hazards
Incompatibility
LEL
Wear protective clothing and SCBA to prevent contact with skin & eyes
Special Fire Fighting Procedures
Stability
Flammable Limits
No data
No data
No data
Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam
Extinguishing Media
Flash Point (Method Used)
Section IV - Physical/Chemical Characteristics
N.D. = No data
Evaporation Rate
(Butyl Acetate = 1)
Melting Point
Specific Gravity (H 0 = 1)
2
Appearance and Odor Chemical bound to paper, no odor
Solubility in Water
Vapor Density (AIR = 1)
Vapor Pressure (mm Hg.)
Boiling Point
Section III - Physical/Chemical Characteristics
% (Optional)
10/05/06
Other Limits
Recommended
Signature of Preparer (optional)
(2,7-Diamino-10-Ethyl-9-Phenylphenanthridinium Bromide)
Ethidium Bromide
Hazardous Components [Specific
Chemical Identity; Common Name(s)]
(301) 251-5990
(301) 251-5990
Telephone Number for information
Emergency Telephone Number
Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must
be marked to indicate that.
Section II - Hazardous Ingredients/Identify Information
InstaStain, Inc.
P.O. Box 1232
West Bethesda, MD 20827
Manufacturer's Name
Section I
InstaStain® Ethidium Bromide
Material Safety Data Sheet
May be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
EDVO-Kit #
Work/Hygienic Practices
Eye Protection
Special
Chemical cartridge respirator with full facepiece.
Local Exhaust
Other Protective Clothing or Equipment
Protective Gloves
Ventilation
Respiratory Protection (Specify Type)
Section VIII - Control Measures
Other Precautions
OSHA Regulation?
Yes
®
IDENTITY (As Used on Label and List)
EDVOTEK
Full size (8.5 x 11”) pdf copy of MSDS available at www.edvotek.com or by request.
Precautions to be Taken in Handling and Storing
N.D.
UEL
Ingestion?
Ingestion: If conscious, give large amounts of water
Steps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Yes
IARC Monographs?
Eyes: Flush with water Inhalation: Move to fresh air
Emergency First Aid Procedures
N.D.
Irritation to upper respiratory tract, skin, eyes
NTP?
Skin?
None
Conditions to Avoid
Medical Conditions Generally Aggravated by Exposure
Signs and Symptoms of Exposure
Carcinogenicity: None identified
None
Yes
X
Inhalation?
Health Hazards (Acute and Chronic)
Route(s) of Entry:
None
Conditions to Avoid
Carbon monoxide, Carbon dioxide
Will Not Occur
May Occur
Section VI - Health Hazard Data
Hazardous
Polymerization
X
None identified
Strong oxidizing agents
Stable
LEL
Wear protective equipment and SCBA with full facepiece
operated in positive pressure mode.
Hazardous Decomposition or Byproducts
Incompatibility
Stability
Section V - Reactivity Data
Flammable Limits
No data
No data
No data
% (Optional)
Use extinguishing media appropriate for surrounding fire.
No data
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Extinguishing Media
Flash Point (Method Used)
Section IV - Physical/Chemical Characteristics
N.D. = No data
Evaporation Rate
(Butyl Acetate = 1)
Melting Point
Specific Gravity (H 0 = 1)
2
Appreciable, (greater than 10%)
No data
No data
No data
Appearance and Odor Clear, liquid, slight vinegar odor
Solubility in Water
Vapor Density (AIR = 1)
Vapor Pressure (mm Hg.)
Boiling Point
Section III - Physical/Chemical Characteristics
Steps to be Taken in case Material is Released for Spilled
Sweep up and place in suitable container for disposal
Other Limits
Recommended
10/05/06
This product contains no hazardous materials as defined by the OSHA Hazard
Communication Standard.
Hazardous Components [Specific
Chemical Identity; Common Name(s)]
Section VII - Precautions for Safe Handling and Use
Treat symptomatically and supportively
OSHA Regulation?
Date Prepared
Signature of Preparer (optional)
(301) 251-5990
Telephone Number for information
(301) 251-5990
Emergency Telephone Number
Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must
be marked to indicate that.
Section II - Hazardous Ingredients/Identify Information
14676 Rothgeb Drive
Rockville, MD 20850
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
Manufacturer's Name
Section I
50x Electrophoresis Buffer
IDENTITY (As Used on Label and List)
Section VII - Precautions for Safe Handling and Use
Emergency First Aid Procedures
Yes
IARC Monographs?
No data available
NTP?
Skin?
None
N.D.
Ingestion: Large amounts may cause diarrhea
Yes
Conditions to Avoid
None
Conditions to Avoid
Medical Conditions Generally Aggravated by Exposure
Signs and Symptoms of Exposure
Carcinogenicity:
Inhalation: No data available
Health Hazards (Acute and Chronic)
Route(s) of Entry:
X
X
None
Section VI - Health Hazard Data
Hazardous
Polymerization
Hazardous Decomposition or Byproducts
Incompatibility
Stability
Section V - Reactivity Data
Unusual Fire and Explosion Hazards
Possible fire hazard when exposed to heat or flame
Special Fire Fighting Procedures
Extinguishing Media Water spray, dry chemical, carbon dioxide, halon or standard foam
Flash Point (Method Used)
N.D.
No data
Evaporation Rate
(Butyl Acetate = 1)
N.D. = No data
No data
No data
Melting Point
White powder, no odor
Insoluble - cold
No data
No data
Specific Gravity (H 0 = 1)
2
Section IV - Physical/Chemical Characteristics
Appearance and Odor
Solubility in Water
Vapor Density (AIR = 1)
Vapor Pressure (mm Hg.)
For 1% solution 194 F
Boiling Point
Section III - Physical/Chemical Characteristics
CAS #9012-36-6
This product contains no hazardous materials as defined by the OSHA Hazard Communication
Standard.
Hazardous Components [Specific
Chemical Identity; Common Name(s)]
Other Limits
Recommended
Signature of Preparer (optional)
Section II - Hazardous Ingredients/Identify Information
14676 Rothgeb Drive
Rockville, MD 20850
(301) 251-5990
Telephone Number for information
(301) 251-5990
Emergency Telephone Number
Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must
be marked to indicate that.
Material Safety Data Sheet
®
May be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
EDVOTEK
Material Safety Data Sheet
May be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
Manufacturer's Name
Section I
IDENTITY (As Used on Label and List)
EDVOTEK
Material Safety Data Sheets
31
314