115

The Biotechnology Education Company
EDVO-Kit
Cancer Gene Detection
115
See Page 3 for storage instructions.
Experiment Objective:
In this experiment, students will gain an understanding of the p53 tumor suppressor gene and its role in familial cancers.
EDVOTEK, Inc. 1-800-EDVOTEK www.edvotek.com
EVT 100202AM
Table of Contents
Experiment Components Experiment Requirements Background Information Experiment Procedures Experiment Overview and General Instructions Agarose Gel Electrophoresis Study Questions 9 11 12 Page 3 3 4
Instructor's Guidelines Notes to the Instructor and Pre-Lab Preparations Experiment Results and Analysis Study Questions and Answers 13 19 20 21 32
Appendices Material Safety Data Sheets
All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.
THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experiment components are
derived from human sources.

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.
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
EVT 100202AM
115
Experiment
Experiment Components
DNA samples are stable at room temperature. However, if the experiment will not be conducted within one month of receipt, it is recommended that the DNA samples be stored in the refrigerator. DNA samples do not require heating prior to gel loading.
Ready-to-Load DNA samples for electrophoresis

A B C D E Standard DNA Fragments Control DNA Patient Peripheral blood DNA Patient Breast Tumor DNA Patient Normal Breast Tissue DNA
Reagents & Supplies

UltraSpec-Agarose powder Concentrated electrophoresis buffer FlashBlue DNA Stain InstaStain Blue cards Practice Gel Loading Solution 1 ml pipet Microtipped Transfer Pipets
Note: If you ordered Experiment #115-Q, the experiment components include InstaStain Ethidium bromide instead of FlashBlue and InstaStain Blue DNA stains.
Requirements
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 Small plastic trays or large weigh boats (for gel destaining) DNA visualization system (white light) Distilled or deionized water
EDVOTEK - The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com FAX: (301) 340-0582 email: info@edvotek.com
EVT 100202AM
115

Experiment
Background Information
About Family Pedigrees
When drawing or studying a family pedigree, the following are general guidelines to the symbols used and their representations:
Female free of cancer Male free of cancer Female with some form of cancer Male with some form of cancer
A Circle represents a female. Female , deceased or A square represents a male. A shaded circle or square refers to a , deceased Male or 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.
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.
Germline Mutation
Hereditary
Somatic Mutation
Sporadic
Cancer gene detection

Many contributory factors have been identified to cause the onset of cancers, that include exposure to certain carcinogens in our diets and environment. Several forms of cancer have familial predispositions. These cancers appear to be linked to inherited mutation of suppressor genes, such as p53. Familial cancers constitute a very small fraction of the total reported cancers and they 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.
Normal Gene Somatic Mutation First Somatic Mutation
Multiple Tumors Bilateral Tumors Early Onset
Second Somatic Mutation
Figure 1: Hereditary and Sporadic models of gene inactivation.
Single Tumors Unilateral Tumors Later onset
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1989,1992,1994,1997,1998, 2000, 2004, 2007, 2009, EDVOTEK, Inc., all rights reserved. EVT 100202AM

Experiment
115
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, 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. With the advent of molecular biology applications to medicine, gene maps and the chromosomal locations of genes are becoming 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 cancerrelated genes, such as p53. Several methods of analysis for the detection of point mutations in genes include DNA sequencing. The Human genome project has provided information to link to the identification of many various cancers and other diseases to DNA sequence information. This information needs to be handled cautiously to assure confidentiality of patients genetic profiles. 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. 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 53,000 molecular nuclear phosphoprotein. Wild type (normal) p53 functions as a cell regulator. There is now well-documented evidence that normal p53 is a sequencespecific 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 Figure 2.
BB.42
BR.36
SS.23 BB.34
CN.2
CN.36
OS.13 BB BR CN LK OS SS Bilateral breast cancer Breast cancer Brain tumor Leukemia Osteosarcoma Soft tissue sarcoma
LK.2
Figure 2: Example of a Li-Fraumeni family pedigree.
115

Experiment
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 mutations are detected in high frequencies. They are between exons 5 through 8 where 95% of the mutations occur. Within this region there five subregions where point mutations are detected in human cancers. The third region of the p53 protein is the carboxyl section that is 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 the mutation and tumor tissue. One such 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 in the individuals with LFS have a single wild type p53 allele. Examination of the p53 has shown a correlation to mutations in the protein as described above.

Experiment
115
Constructing the 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 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, Jane, aged 2 who is Patrick's sister was diagnosed with childhood leukemia and subsequently died. Patrick's two other brothers, Robert, 28 and Curtis, 30, are in good health and free of cancer. Valerie's sister, Nancy is free of cancer. Nancy's son, Michael was diagnosed at the age of 3 as having sarcoma. Recently, at the age of 18, he was diagnosed as having osteosarcoma. Nancy's other son, John, and daughter, Jessica, are free of cancer. Valerie has five children: Justin (16), Sheila (14), Robert (10), Angela (8), and Anthony (6), none of whom show any signs of cancer at this time. She was interested in the p53 diagnostic test to determine if she inherited mutations.
115

Experiment
Constructing the Family Pedigree

The familial pedigree 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 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 the simulation experiment which follows, 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 the palindrome CAGCTG. A restriction enzyme was used as a probe to cut the simulated amplified gene for Valeries 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 DNA fragment banding pattern. The DNA obtained from blood lymphocyte 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 samples with the control wild type and DNA markers will be separated by agarose gel electrophoresis and stained.

Experiment
115
Experiment Overview and General Instructions

Experiment Objective:
In this experiment, students will gain an understanding of the p53 tumor suppressor gene and its role in familial cancers.
LABORATORY SAFETY
1. Gloves and goggles should be worn routinely as good laboratory practice.
Experiment Procedure
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.
115

Experiment
Experiment Overview: Flow Chart
1 2
Remove end blocks & comb, then submerge gel under buffer in electrophoresis chamber
Prepare agarose gel in casting tray
Load each sample in consecutive wells
Attach safety cover,connect leads to power source and conduct electrophoresis
After electrophoresis, transfer gel for staining
5
(-) 1 2 3 4 5 6
FlashBlue DNA stain
6
Analysis on white light source
(+)
Gel pattern will vary depending upon experiment.
10

Experiment
115
Agarose Gel Electrophoresis

Prepare the Gel
1. Prepare an agarose gel with specifications summarized below. Your instructor will specify which DNA stain you will be using. Agarose gel concentration required: 0.8% Recommended gel size: Number of sample wells required: 7 x 7 cm or 7 x 14 cm (two gels) 5
Wear Gloves & goggles
For gels to be stained with FlashBlue or InstaStain Blue, prepare gels according to Appendix A. For gels to be stained with InstaStain Ethidium bromide, prepare gels according to Appendix B. Step-by-step guidelines for agarose gel preparation are summarized in Appendix D.
Placement of well-former template: first set of notches ( 7 x 7 cm) first & third set of notches (7 x 14 cm)
Load the Samples

2. Load the DNA samples in tubes A - E into the wells in consecutive order. For gels to be stained with FlashBlue or InstaStain Blue, fill wells with 35 - 38 l. For gels to be stained with InstaStain Ethidium Bromide, fill wells with 18 - 20 l. Lane 1 2 3 4 5 Tube A B C D E Standard DNA Fragments Control DNA Patient Peripheral blood DNA Patient Breast Tumor DNA Patient Normal Breast Tissue DNA
Reminders:
During electrophoresis, the DNA samples migrate through the agarose gel towards the positive electrode. Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.
Black Sample wells
Run the Gel

3. After DNA samples are loaded, connect the apparatus to the D.C. power source and set the power source at the required voltage. 4. Check that current is flowing properly - you should see bubbles forming on the two platinum electrodes. Conduct electrophoresis for the length of time specified by your instructor. 5. After electrophoresis is completed, proceed to DNA staining and visualization. Refer to Appendix E, F, G, or H for the appropriate staining instructions. 6. Document the results of the gel by photodocumentation.
Red
Alternatively, place transparency film on the gel and trace it with a permanent marking pen. Remember to include the outline of the gel and the sample wells in addition to the migration pattern of the DNA bands.
11
32
Material Safety Data Sheet
EDVOTEK
May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements.
EDVOTEK

EDVOTEK
IDENTITY (As Used on Label and List)
Practice Gel Loading Solution

50x Electrophoresis Buffer
Agarose
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 I
Section I
(301) 251-5990
Manufacturer's Name Emergency Telephone Number
Manufacturer's Name
Emergency Telephone Number
Section I
EDVOTEK, Inc.
Address (Number, Street, City, State, Zip Code)
Telephone Number for information
EDVOTEK, Inc.
(301) 251-5990
EDVOTEK, Inc.
(301) 251-5990
(301) 251-5990
(301) 251-5990
(301) 251-5990 14676 Rothgeb Drive Rockville, MD 20850
10/05/06
Signature of Preparer (optional) Date Prepared
Experiment
115
14676 Rothgeb Drive Rockville, MD 20850

10/05/06
10/05/06
Signature of Preparer (optional)
Date Prepared
14676 Rothgeb Drive Rockville, MD 20850 Section II - Hazardous Ingredients/Identify Information

Section II - Hazardous Ingredients/Identify Information
Other Limits Recommended % (Optional)
OSHA PEL ACGIH TLV Hazardous Components [Specific Chemical Identity; Common Name(s)] Other Limits Recommended
Date Prepared

Other Limits Recommended % (Optional)
Hazardous Components [Specific Chemical Identity; Common Name(s)]
OSHA PEL
ACGIH TLV
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
% (Optional)
This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard.
CAS #9012-36-6
Section III - Physical/Chemical Characteristics

No data
Specific Gravity (H 0 = 1) 2 Boiling Point

No data
For 1% solution 194 F No data No data
Boiling Point Vapor Pressure (mm Hg.) Vapor Density (AIR = 1) Solubility in Water Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (Butyl Acetate = 1)
Boiling Point
No data No data
Vapor Pressure (mm Hg.) Melting Point Evaporation Rate (Butyl Acetate = 1) Vapor Density (AIR = 1) Solubility in Water
Specific Gravity (H 0 = 1) 2
No data No data No data Appreciable, (greater than 10%)

Appearance and Odor
No data No data No data
Vapor Pressure (mm Hg.)
No data No data
Insoluble - cold White powder, no odor
Melting Point
No data No data
Vapor Density (AIR = 1)
No data
Evaporation Rate (Butyl Acetate = 1)
Solubility in Water
Soluble
Appearance and Odor Clear, liquid, slight vinegar odor
Appearance and Odor
Blue liquid, no odor
Section IV - Physical/Chemical Characteristics

LEL UEL

Flash Point (Method Used)
N.D. = No data
LEL UEL

N.D.
No data
N.D. = No data
Flammable Limits LEL
Flammable Limits
No data Use extinguishing media appropriate for surrounding fire. Wear protective equipment and SCBA with full facepiece operated in positive pressure mode. None identified
Flammable Limits
N.D.
N.D.
UEL
N.D.
Extinguishing Media
Dry chemical, carbon dioxide, water spray or foam

Extinguishing Media Special Fire Fighting Procedures
Extinguishing Media Water spray, dry chemical, carbon dioxide, halon or standard foam Special Fire Fighting Procedures
Special Fire Fighting Procedures
Use agents suitable for type of surrounding fire. Keep upwind, avoid breathing hazardous sulfur oxides and bromides. Wear SCBA.
Unusual Fire and Explosion Hazards
Possible fire hazard when exposed to heat or flame

Unknown
None
Section V - Reactivity Data

Stability Stable
Incompatibility
Stability
Unstable
Conditions to Avoid
Unstable
Conditions to Avoid

Stability
Incompatibility
Stable
X
Strong oxidizing agents Carbon monoxide, Carbon dioxide
Conditions to Avoid
None
None
Unstable Stable
Conditions to Avoid
Incompatibility
None
Hazardous Decomposition or Byproducts
X No data available
None
Sulfur oxides, and bromides

Hazardous Polymerization May Occur Will Not Occur Inhalation? Skin?
Hazardous Polymerization
May Occur
Conditions to Avoid
Will Not Occur
None
None
Ingestion?
Section VI - Health Hazard Data

Yes
Ingestion? Yes
Hazardous Polymerization Route(s) of Entry:
May Occur Will Not Occur
Conditions to Avoid
None
Route(s) of Entry:
Inhalation?
Yes
Skin?
Route(s) of Entry:

Yes
Inhalation? Health Hazards (Acute and Chronic)
Yes
Yes
Health Hazards (Acute and Chronic)
Acute eye contact: May cause irritation. No data available for other routes.
Yes
OSHA Regulation?
Skin?
Yes Inhalation: No data available

Carcinogenicity: NTP? Signs and Symptoms of Exposure
Ingestion?
Yes Ingestion: Large amounts may cause diarrhea

IARC Monographs? OSHA Regulation?
None
NTP? IARC Monographs?
Carcinogenicity: OSHA Regulation?
No data available
Signs and Symptoms of Exposure Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures
NTP?
IARC Monographs?
Carcinogenicity: None identified
Material Safety Data Sheets
Signs and Symptoms of Exposure
May cause skin or eye irritation
Irritation to upper respiratory tract, skin, eyes None
No data available
Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures
Medical Conditions Generally Aggravated by Exposure None reported
Emergency First Aid Procedures
No data available
Treat symptomatically and supportively. Rinse contacted area with copious amounts of water.
Ingestion: If conscious, give large amounts of water Skin: Wash with soap and water
Eyes: Flush with water Inhalation: Move to fresh air
Treat symptomatically and supportively
Section VII - Precautions for Safe Handling and Use

Steps to be Taken in case Material is Released for Spilled

Wear eye and skin protection and mop spill area. Rinse with water.
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.
Sweep up and place in suitable container for disposal

Observe all federal, state, and local regulations.
Dispose in accordance with all applicable federal, state, and local enviromental regulations. Avoid eye and skin contact.
Normal solid waste disposal

Precautions to be Taken in Handling and Storing
Avoid eye and skin contact.

Other Precautions
None
Other Precautions
None
None
Other Precautions
None
Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
Section VIII - Control Measures

Special

Respiratory Protection (Specify Type)

Chemical cartridge respirator with full facepiece.

Ventilation Protective Gloves Local Exhaust
Ventilation Other
Local Exhaust
Yes None Splash proof goggles
None
Mechanical (General)
Yes
Yes
Special
Ventilation
Local Exhaust
Special
Yes Yes
Other Protective Clothing or Equipment None Work/Hygienic Practices
Other Eye Protection
None None _Safety goggles
Protective Gloves
Yes
Eye Protection
Mechanical Gen. dilution ventilation Protective Gloves
Other
Yes
Other Protective Clothing or Equipment
Eye Protection
Splash proof goggles Impervious clothing to prevent skin contact
Other Protective Clothing or Equipment
None required
Work/Hygienic Practices
Avoid eye and skin contact
None
Work/Hygienic Practices
None
EDVOTEK
EDVOTEK
InstaStain Blue, FlashBlue
InstaStain Ethidium Bromide
Section I
Section I
EDVOTEK, Inc.
(301) 251-5990
InstaStain, Inc. P.O. Box 1232 West Bethesda, MD 20827 (301) 251-5990
Date Prepared
Manufacturer's Name
Emergency Telephone Number
(301) 251-5990
(301) 251-5990
10/05/06
14676 Rothgeb Drive Rockville, MD 20850

03-26-09
Date Prepared

Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Other Limits Recommended % (Optional)
OSHA PEL ACGIH TLV Hazardous Components [Specific Chemical Identity; Common Name(s)] Other Limits Recommended

% (Optional)
Ethidium Bromide (2,7-Diamino-10-Ethyl-9-Phenylphenanthridinium Bromide) CAS# 139-33-3
Data not available
Methylene Blue 3.7 Bis (Dimethylamino) Phenothiazin 5 IUM Chloride CAS # 61-73-4 No data available

Boiling Point

Solubility in Water Vapor Density (AIR = 1) Vapor Pressure (mm Hg.) Boiling Point
No data No data No data Soluble - cold

Soluble
Evaporation Rate (Butyl Acetate = 1) Melting Point
Specific Gravity (H 0 = 1) 2
Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (Butyl Acetate = 1)
Vapor Pressure (mm Hg.) Vapor Density (AIR = 1) Solubility in Water Appearance and Odor Chemical bound to paper, no odor
Appearance and Odor Chemical bound to paper, no odor

Flash Point (Method Used) Flammable Limits LEL UEL
Flash Point (Method Used) Extinguishing Media

No data No data
No data
N.D. = No data
Flammable Limits LEL UEL
No data available
Extinguishing Media
N.D.
N.D.
Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam
Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam Wear protective clothing and SCBA to prevent contact with skin & eyes
Self contained breathing apparatus and protective clothing to prevent contact with skin and eyes Emits toxid fumes under fire conditions
Emits toxic fumes

Stability Stable
Incompatibility

Conditions to Avoid
Unstable
Stability
Unstable Stable
Incompatibility
Conditions to Avoid
None
X
Strong oxidizing agents
None
Strong oxidizing agents Toxic fumes of Carbon monoxide, Carbon dioxide, nitrogen oxides, sulfur oxides, hydrogen, chloride gas
Conditions to Avoid
Material Safety Data Sheets
Carbon monoxide, Carbon dioxide, nitrogen oxides, hydrogen bromide gas Hazardous Polymerization Route(s) of Entry: May Occur Will Not Occur
Conditions to Avoid
Hazardous Polymerization May Occur Will Not Occur Inhalation? Skin?
None
Ingestion?
None

Route(s) of Entry:

Yes Yes Yes Inhalation: Cyanosis
OSHA Regulation?
Inhalation? Yes Skin?
Yes
Ingestion? Yes Health Hazards (Acute and Chronic) Chronic: May alter genetic material Acute: Material irritating to mucous membranes, upper respiratory tract, eyes, skin Carcinogenicity: No data available NTP? Signs and Symptoms of Exposure IARC Monographs? OSHA Regulation?
Skin: May cause skin irritation

NTP?
Eyes: May cause eye irritation

IARC Monographs?
Carcinogenicity: Signs and Symptoms of Exposure
Meets criteria for proposed OSHA medical records rule PEREAC 47.30420.82 No data available
Irritation to mucous membranes and upper respiratory tract

Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures
Medical Conditions Generally Aggravated by Exposure No data available Emergency First Aid Procedures
No data Treat symptomatically and supportively
Treat symptomatically


Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
Ventilate area and wash spill site

Wear SCBA, rubber boots, rubber gloves
Mix material with a combustible solvent and burn in chemical
incinerator equipped with afterburner and scrubber. Check local and state regulations.
Mix material with combustible solvent and burn in a chemical incinerator equipped afterburner and scrubber
Keep tightly closed. Store in cool, dry place

Other Precautions
Other Precautions
Use in chemical fume hood with proper protective lab gear. Mutagen
None

Respiratory Protection (Specify Type) Ventilation Protective Gloves

MIOSH/OSHA approved, SCBA
Local Exhaust Mechanical (General)
Special
Respiratory Protection (Specify Type) Ventilation Local Exhaust
SCBA Yes
Special
Experiment
Chem. fume hood
115
Required Rubber
Other Protective Clothing or Equipment Work/Hygienic Practices
Other Eye Protection
No
Other
None
Chem. safety goggles Rubber boots
Protective Gloves
Rubber
Other Protective Clothing or Equipment Work/Hygienic Practices
Eye Protection
Chem. safety goggles Rubber boots Use in chemical fume hood with proper protective lab gear.
33
EDVOTEK Series 100 Electrophoresis Experiments:

Cat. # Title
101 102 103 104 105 109 112 114 115 116 117 118 124 130
Principles and Practice of Agarose Gel Electrophoresis Restriction Enzyme Cleavage Patterns of DNA PCR - Polymerase Chain Reaction Size Determination of DNA Restriction Fragments Mapping of Restriction Sites on Plasmid DNA DNA Fingerprinting - Identification of DNA by Restriction Fragmentation Patterns Analysis of Eco RI Cleavage Patterns of Lambda DNA DNA Paternity Testing Simulation Cancer Gene Detection Sickle Cell Gene Detection (DNA-based) Detection of Mad Cow Disease Cholesterol Diagnostiics DNA-based Screening for Smallpox DNA Fingerprinting - Amplification of DNA for Fingerprinting
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Visit our web site for information about the above experiments and other products in EDVOTEKs comprehensive offerings for biotechnology and biology education.
34
EVT 100202AM

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The Biotechnology Education Company

Cancer Gene Detection

See Page 3 for storage instructions.

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

Cancer Gene Detection

Appendices Material Safety Data Sheets

derived from human sources.

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

Cancer Gene Detection

Ready-to-Load DNA samples for electrophoresis

Reagents & Supplies

Cancer Gene Detection

Cancer gene detection

Normal Gene Somatic Mutation First Somatic Mutation

Multiple Tumors Bilateral Tumors Early Onset

Second Somatic Mutation

Figure 1: Hereditary and Sporadic models of gene inactivation.

Single Tumors Unilateral Tumors Later onset

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

Cancer Gene Detection

Figure 2: Example of a Li-Fraumeni family pedigree.

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

Cancer Gene Detection

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

Cancer Gene Detection

Constructing the Family Pedigree

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

Cancer Gene Detection

Constructing the Family Pedigree

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

Cancer Gene Detection

Experiment Overview and General Instructions

Laboratory notebook recordings:

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

Cancer Gene Detection

Experiment Overview: Flow Chart

Prepare agarose gel in casting tray

Load each sample in consecutive wells

Attach safety cover,connect leads to power source and conduct electrophoresis

After electrophoresis, transfer gel for staining

FlashBlue DNA stain

Gel pattern will vary depending upon experiment.

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Cancer Gene Detection

Agarose Gel Electrophoresis

Wear Gloves & goggles

Load the Samples

Run the Gel

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Material Safety Data Sheet

IDENTITY (As Used on Label and List)

Practice Gel Loading Solution

IDENTITY (As Used on Label and List)

Emergency Telephone Number

Address (Number, Street, City, State, Zip Code)

Telephone Number for information

14676 Rothgeb Drive Rockville, MD 20850

Signature of Preparer (optional)

14676 Rothgeb Drive Rockville, MD 20850 Section II - Hazardous Ingredients/Identify Information

Section II - Hazardous Ingredients/Identify Information

Hazardous Components [Specific Chemical Identity; Common Name(s)]

Section III - Physical/Chemical Characteristics

Section III - Physical/Chemical Characteristics