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EDVO-Kit #

109

& 109-Q

DNA Fingerprinting -

Identification of DNA Restriction Fragmentation Patterns Storage: See Page 3 for specific storage instructions

Experiment Objective: The objective of this experiment is to develop a basic understanding of DNA fingerprinting. Variations in restriction enzyme cleavage patterns obtained from different DNA molecules will be analyzed and the possible perpetrator of a crime will be identified using the logic of DNA fingerprinting.

This document includes instructions for both EDVOTEK Experiment # 109 and # 109-Q. Please follow instructions for the appropriate experiment. Experiment #109 is designed for DNA staining with InstaStain® Methylene Blue. Experiment # 109-Q is designed for DNA staining with InstaStain® Ethidium Bromide.

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EDVO-Kit #

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

109 109 Table of Contents This document includes instructions for both EDVOTEK Experiment # 109 and # 109-Q. Please follow instructions for the appropriate experiment. Experiment # 109 is designed for DNA staining with InstaStain® Methylene Blue. Experiment # 109-Q is designed for DNA staining with InstaStain® Ethidium Bromide.

Page

Experiment Components Experiment Requirements Background Information Experiment Procedures Experiment Overview and General Instructions Agarose Gel Electrophoresis Agarose Gel Requirements for this Experiment Preparing the Gel Bed Casting Agarose Gels Preparing the Gel for Electrophoresis Loading the Samples Running the Gel Staining and visualization of DNA Study Questions Instructor's Guidelines Notes to the Instructor Pre-Lab Preparations Experiment Results and Analysis Study Questions and Answers

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

Appendices 0.8% Agarose Gel Preparation A DNA Staining with InstaStain® Methylene Blue B DNA Staining with InstaStain® Ethidium Bromide C Quantity Preparations for Agarose Gel Electrophoresis Staining and Visualization of DNA D Method 1: InstaStain® MetBlue One-step Staining and destaining E Method 2: InstaStain® MetBlue Cards F Method 3: Liquid Methylene Blue Plus™ G Method 4: InstaStain® Ethidium Bromide Cards

Material Safety Data Sheets

of the experiment components are derived from human sources.

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EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.

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3 3 5

12 15 15 15 16 17 18 18 19 21 22 25 27 28

30 31 32

33 34 36 37 38

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

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109 Components & Requirements 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. Over time, some evaporation of samples may occur. Before distributing reagents to students, check sample volumes as described in the Instructor’s Pre-Lab Preparation section.

Ready-to-Load™ DNA samples for electrophoresis

A B C D E F

DNA from crime scene cut with Enzyme 1 DNA from crime scene cut with Enzyme 2 DNA from Suspect 1 cut with Enzyme 1 DNA from Suspect 1 cut with Enzyme 2 DNA from Suspect 2 cut with Enzyme 1 DNA from Suspect 2 cut with Enzyme 2

Store entire experiment at room temperature. DNA samples do not require heating prior to gel loading.

Reagents & Supplies • • • • • • • •

UltraSpec-Agarose™ powder Concentrated electrophoresis buffer Practice Gel Loading Solution 1 ml pipet 100 ml graduated cylinder (packaging for samples) Microtipped Transfer Pipets DNA Stain for Standard Series 100 experiments • InstaStain® Methylene Blue • Methylene Blue Plus™ DNA Stain for Series 100-Q experiments • InstaStain® Ethidium Bromide

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 Distilled or deionized water For gel staining with InstaStain® Methylene Blue • Small plastic trays or large weigh boats for destaining • White light DNA visualization system For gel staining with InstaStain® Ethidium Bromide • UV Transilluminator Photodocumentation system (optional)

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DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

EDVO-Kit #

109 109 This document includes instructions for both EDVOTEK Experiment # 109 and # 109-Q. instructions for the appropriate experiment.

Please follow

Experiment #109 Designed for DNA staining and visualization with InstaStain® Methylene Blue. This experiment requires a 0.8% gel with the following volume:

30 ml (7 x 7 cm)

or

60 ml (7 x 14 cm)

Refer to Table A.1 or A.2 in Appendix A for agarose gel preparation specifications.

Experiment # 109-Q Designed for DNA staining and visualization with InstaStain® Ethidium Bromide. This experiment requires a 0.8% gel with the following volume:

25 ml (7 x 7 cm)

or

50 ml (7 x 14 cm)

Refer to Table A.3 or A.4 in Appendix B for agarose gel preparation specifications.

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DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

EDVO-Kit #



109 Introduction to DNA Fingerprinting

Human DNA fingerprinting was pioneered by Dr. Alex Jeffreys at the University of Leicester in 1984. His analytical method led to the apprehension of a murderer in the first DNA fingerprinting conviction in September 1987 in the UK. Two months later, the first U.S. conviction based on DNA fingerprinting occurred in Orlando, Florida. Since then, the use of DNA fingerprinting has led to thousands of criminal convictions, as well as dozens of exonerations. In contrast to earlier methodologies, such as blood typing which can only exclude a suspect, DNA fingerprinting can provide positive identification with great accuracy. In addition to criminal identification cases, DNA fingerprinting is now used routinely in paternity determinations and for the identification of genetic disease "markers". It is also used for the identification of human remains, such as in war casualties, and was used extensively to identify victims of the September 11, 2001 terrorist attacks on the World Trade Center, the Pentagon, and the plane which crashed in a field near Shanksville, Pennsylvania. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Human cells contain two types of DNA. The first type is cellular chromosomal DNA, which is packaged in 23 sets of chromosomes in the nucleus of the cell. This DNA, obtained from both parents, reflects the combined parental genetic inheritance of an individual. DNA fingerprinting utilizing cellular DNA involves analysis of the sequence of two alleles for a particular gene. The second type of DNA is different from cellular DNA and is present only in the mitochondria, which are the energy-producing organelles of the cell. Mitochondrial DNA is inherited maternally by both males and females and is extremely useful in the analysis of specific cases where fraternal linkages are important to determine. For example, a brother, sister, half brother or half sister who share the same mother would inherit the same mitochondrial DNA. Identification is determined by sequencing certain regions within mitochondrial DNA, which is a single circular chromosome composed of 16,569 base pairs. DNA fingerprinting developed by Dr. Jeffreys utilizes cellular chromosomal DNA submitted to restriction enzyme digestion and Southern blot analysis. When human DNA is digested by a restriction enzyme, a very large number

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Background Information

DNA typing (also called DNA profile analysis or DNA fingerprinting) is the process whereby the genomic DNA of an organism is analyzed by examining several specific, variable DNA sequences located throughout the genome. In humans, DNA fingerprinting is now used routinely for identification purposes.



DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

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109

of DNA fragments are generated. When separated by agarose gel electrophoresis, the numerous DNA fragments appear as a "smear" on the gel. Labeled probes are used to detect Restriction Fragment Length Polymorphic (RFLP) regions within DNA, which will be described in greater detail. The RFLP method is statistically very accurate but requires relatively large amounts of DNA and takes several weeks to perform. = Separation of 2 DNA strands = Primer 1 = Primer 2

Target Sequence 5' 3'

3' 5'

5'

3' Denature 94°C

Cycle 1

3'

5'

5'

Cycle 2

3'

3'

3'

3'

5' 5'

3'

Cycle 3

Background Information

Introduction to DNA Fingerprinting

5'

5'

5'

5' 3' 5'

5'

5' 3' 5' 3'

5' 3' 5'

5' 3' 5'

5'

5'

5'

5' 5'

5'

5' 5'

5'

Anneal 2 primers 45°C

5' 3' 5'

5' 3' 5'

5' 3' 5' 3' 5' 3' 5'

3'

Extension 72°C

3'

3'

Figure 1: The Polymerase Chain Reaction

In recent years, the use of the RFLP method has been overtaken by the Polymerase Chain Reaction (PCR) method because of two important advantages. The first is the sensitivity of PCR, which allows for DNA fingerprinting identification using much smaller amounts of DNA. This is because PCR is able to amplify DNA to facilitate analysis. A second advantage is the speed of PCR analysis, which allows critical questions to be answered more quickly compared to Southern Blot analysis. Many important concepts of molecular biology can be conveyed in the context of DNA Fingerprinting methods. In this experiment, emphasis is placed on concepts related to RFLP analysis. The experiment activities will focus on the identification of DNA by analyzing restriction fragmentation patterns separated by agarose gel 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 © 1989,1992,1994,1997,1998, 2000, 2004, 2007, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

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109 Introduction to DNA Fingerprinting Use of Restriction Enzymes in DNA Fingerprinting

The two most commonly used restriction enzymes for DNA profile analysis are Hae III and Hinf I, which are 4-base and 5-base cutting enzymes. The examples in the figure 2 show recognition sites for various restriction enzymes.

Bam HI

Hae III

Pst I

Hinf I

↓ 5'....GGATCC....3' 3'....CCTAGG....5' ↑ ↓ 5'....CTGCAG....3' 3'....GACGTC....5' ↑

↓ 5'....GGCC....3' 3'....CCGG....5' ↑ ↓ 5'....GANTC....3' 3'....CTNAG....5' ↑

The size of the DNA fragments generated depends on the distance between the recognition sites. In general, the longer the DNA molecule, the greater the probability that a given recognition site will occur. Human DNA is very large and contains approximately three billion base pairs. A restriction enzyme having a 6‑base pair recognition site, such as Eco RI, would be expected to cut human DNA into approximately 750,000 different fragments.

DNA is highly polymorphic - that is, no two individuals have exactly the same pattern of Figure 2: Restriction enzyme recognition sites restriction enzyme recognition sites in their DNAs. A large number of alleles exist in the population. Alleles, which are alternate forms of a gene, result in alternative expressions of genetic traits which can be dominant or recessive. Chromosomes occur in matching pairs, one of maternal and the other of paternal origin. The two copies of a gene (alleles) at a given chromosomal locus represent a composite of the parental genes constituting an individual’s unique genotype. It follows that alleles have differences in their base sequences which consequently creates differences in the distribution and frequencies of restriction enzyme recognition sites. Other differences in base sequences between individuals can occur because of mutations and deletions. Such changes can also create or eliminate a recognition site. Polymorphic DNA refers to chromosomal regions that vary widely from individual to individual. By examining several of these regions within the genomic DNA obtained from an individual, one may obtain a "DNA fingerprint" for that individual. The most commonly used polymorphisms are

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Background Information

DNA fingerprinting involves the electrophoretic analysis of DNA fragment sizes generated by restriction enzymes. Restriction enzymes are endonucleases which catalyze the cleavage of phosphodiester bonds within both strands of DNA. The points of cleavage occur in or near very specific palindromic sequences of bases called recognition sites, which are generally 4 to 8 base pairs in length.



DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

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109 Introduction to DNA Fingerprinting

Background Information

those which vary in length; these are known as Fragment Length Polymorphisms (FLPs). There are two main reasons for the occurrence of FLPs. Restriction Fragment Length Polymorphisms (RFLPs) are the result of variations in length of a given segment of genomic DNA between two restriction endonuclease recognition sites among individuals of the same species. RFLPs are the result of an altered restriction enzyme cut site that may be the result of a mutation of a restriction enzyme recognition site. A second major type of FLP occurs mainly in "intergenic" or noncoding regions of DNA and is known as Variable Number of Tandem Repeats (VNTRs). In this case, segments of DNA that contain sequences from 2 to 40 bases in length repeat in tandem manner many times. The number of segments or "core units" repeats varies among individuals of the same species. The restriction enzyme cut sites are not altered. VNTR loci are very polymorphic. There are potentially hundreds of alleles at a single locus and therefore they are very useful in DNA fingerprinting. Ten to fifteen percent of mammalian DNA consists of sets of repeated, short sequences of bases that are tandemly

Probe

Allele 1 2.0 Kb Probe

Allele 2 1.5 Kb

1

0.5 Kb

Genotypes 2 3 4

An allele is recognized by a probe that spans over the internal restriction enzyme site which is present in certain alleles. In Allele 1, the internal restriction enzyme site is missing. In Allele 2, the internal restriction enzyme is present resulting in two fragments. Upon separation by agarose gel electrophoresis of the digested Allele 2, two fragments are generated. Probes will bind to the 2.0 Kb allele as well as the two smaller fragments (1.5 Kb and 0.5 Kb) generated by the restriction enzyme digestion because the probe spans over the two fragments as well as the intact allele. Lane 1 DNA Marker Lane 2 Homozygous allele Lane 3 Heterozygous alleles where one can be cut with the restriction enzyme Lane 4 Homozygous alleles where both are cut with the restriction enzyme

Figure 3: RFLP as Determined in Southern Blot analysis

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

EDVO-Kit #



109 Introduction to DNA Fingerprinting

Probe

A Probe

B 40 Kb Probe

C 50 Kb Genotypes 2 3 4 5 6

The DNA Fingerprinting Process 7 50 Kb C 40 Kb B 30 Kb A

Probe overlaps both the variable region, as well as adjacent part of the genome. Arrows show restriction enzyme sites with probe for Southern Blot analysis. PCR can also be used to detect variable nucleotide regions.

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7

When these arrays are flanked by recognition sites, the length of the repeat will determine the size of the restriction enzyme fragment generated. There are several types of these short, repetitive sequences and they have been characterized.

DNA Marker Homozygous Copies Heterozygous VNTR Heterozygous VNTR Homozygous Copies Heterozygous VNTR Homozygous Copies

Agarose gel electrophoresis is a procedure used to analyze DNA fragments generated by restriction enzymes. The gel consists of microscopic pores that act as a molecular sieve. Samples of DNA are loaded into wells made in the gel during casting. Since DNA has a negative charge at neutral pH, it migrates through the gel towards the positive electrode during electrophoresis. DNA fragments are separated by the gel according to their size. The smaller the fragment the faster it migrates. After electrophoresis, the DNA can be visualized by staining the gel with dyes. Restriction enzyme cleavage of relatively small DNA molecules, such as plasmids and viral DNAs, usually results in discrete banding patterns of the DNA fragments after electrophoresis. However, cleavage of large and complex DNA, such as human chromosomal DNA, generates so many differently sized fragments that the resolving capacity of the gel is exceeded. Consequently, the cleaved DNA is visualized as a smear after staining and has no obvious banding patterns.

(Lanes 3, 4, and 6 represent different combinations of the three VNTRs.) Figure 4: VNTR Demonstrating Variable Numbers of Nucleotide Tandem Regions

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Background Information

TGTTTA|TGTTTA|TGTTTA|.........variable number

30 Kb

1

arranged in arrays. The length of these arrays (the amount of repeated sets) varies between individuals at different chromosomal loci.

10

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109 Introduction to DNA Fingerprinting

Background Information

RFLP analysis of genomic DNA is facilitated by Southern Blot analysis. After electrophoresis, the DNA fragments in the gel are denatured by soaking in an alkali solution. This causes double-stranded DNA fragments to be converted into single-stranded form (no longer base-paired in a double helix). A replica of the electrophoretic pattern of DNA fragments in the gel is made by transferring (blotting) them to a sheet of nylon membrane. This is done by placing the membrane on the gel after electrophoresis and transferring the fragments to the membrane by capillary action or suction by vacuum. The DNA, which is not visible, becomes permanently adsorbed to the membrane, and can be manipulated easier than gels.

2

3

4

Analysis of the blotted DNA is done by hybridization with a labeled DNA probe. In forensic RFLP analysis, the probe is a DNA fragment that contains base sequences which are complementary to the variable arrays of tandemly repeated 1 Figure 5: DNA Fingerprinting sequences found in by RFLP Analysis the human chromoEvidence somes. Probes can be Suspect's labeled with isotoBlood pic or non-isotopic 8 reporter molecules, such as fluorescent dyes used for detec1. Collection of DNA tion. A solution 2. Extraction of DNA containing the single3. DNA cut into fragments by restriction stranded probe is enzymes incubated with the 4. DNA fragments separated by agarose membrane containgel electrophoresis 7 Autoradiograph ing the blotted, 5. DNA denatured into single strands 6. Blot DNA onto a nylon membrane single-stranded (de(Southern Blot) natured) DNA frag7. Nylon membrane soaked with ments. Under the probes that bind to target DNA proper conditions, fragments and detected. the probe will only 8. Computer analysis base pair (hybridize) to those fragments 6 containing the complementary repeated sequences. The 5 Southern Blot membrane is then washed to remove excess probe. If the

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

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109 Introduction to DNA Fingerprinting

In forensic cases, DNA samples can be extracted and purified from small specimens of skin, blood, semen, or hair roots collected at the crime scene. DNA that is suitable for analysis can also be obtained from dried stains of semen and blood. The RFLP analyses performed on these samples is then compared to samples obtained from the suspect. If the RFLP patterns match, it is then beyond reasonable doubt that the suspect was at the crime scene. In practice, several different probes containing different types of repetitious sequences are used in the hybridizations in order to satisfy certain statistical criteria for absolute, positive identification. The use of different restriction enzymes allow for accuracies in positive identifications of greater than one in 100 million. The Polymerase Chain Reaction (PCR) method amplifies target sequences of DNA, which are referred to as AMRFLPs. PCR has made it possible for very small amounts of DNA found at crime scenes to be amplified for DNA fingerprinting analysis. A specific set of two primers is used to prime DNA polymerase to synthesize many copies of the targeted areas of DNA. In this experiment, DNAs are digested by restriction enzymes and the fragmentation patterns serve as the individual fingerprint. The DNA fragmentation patterns can be analyzed directly in the stained agarose gel, which eliminates the need for a Southern blot. In this hypothetical case, DNA obtained from two suspects are cleaved with two restriction enzymes in separate reactions. The objective is to analyze and match the DNA fragmentation patterns after agarose gel electrophoresis and determine if Suspect 1 or Suspect 2 was at the crime scene. THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA.

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Background Information

probe is isotopically labeled to the membrane, it is then placed on an x-ray film for several hours. This process is known as autoradiography. Only DNA fragments that have hybridized to the probe will reveal their positions on the film because the localized areas of radioactivity cause exposure. The hybridized fragments appear as discrete bands (fingerprint) on the film and are in the same relative positions as they were in the agarose gel after electrophoresis. Only specific DNA fragments, of the hundreds of thousands of fragments present, will hybridize with the probe because of the selective nature of the hybridization (base pairing) process.

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DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

109 109

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DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

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109 Experiment Overview and General Instructions Experiment Objective: The objective of this experiment is to develop a basic understanding of DNA fingerprinting. Variations in restriction enzyme cleavage patterns obtained from different DNA molecules will be analyzed and the possible perpetrator of a crime will be identified using the logic of DNA fingerprinting.

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.

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

The Experiment

LABORATORY SAFETY

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DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

109 Experiment Overview and General Instructions Experiment Overview: Flow chart

The Experiment

Prepare agarose gel in casting tray Remove end blocks, comb and submerge gel under buffer in electrophoresis chamber

DNA Samples

Load each sample in consecutive wells.

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

100 Series

After electrophoresis, transfer gel for staining

(Standard)

© 2006 EDVOTEK, Inc.

InstaStain® Methylene Blue

Analysis on White light source After destaining

100-Q Series

InstaStain® Ethidium Bromide

Analysis on UV Transilluminator No destaining

Gel pattern will vary depending on experiment

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

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109 Experiment Overview and General Instructions About the Electrophoresis Samples Samples in EDVOTEK Series 100, 100-Q and Sci-On® Series electrophoresis experiments are packaged in individual 1.5 ml or 0.5 ml microtest tubes. Individual 1.5 ml or 0.5 ml microtest tubes •

Your instructor may have aliquoted samples into a set of tubes for each lab group. Alternatively, you may be required to withdraw the appropriate amount of sample from the experiment stock tubes.



•

Check the sample volume. Sometimes a small amount of sample will cling to the walls of the tubes. Make sure the entire volume of sample is at the bottom of the tubes before starting to load the gel.



•

Briefly centrifuge the sample tubes, or tap each tube on the tabletop to get all the sample to the bottom of the tube.

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

The Experiment



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DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

109 Agarose Gel Electrophoresis

The Experiment

Agarose Gel Requirements for this Experiment

•

Recommended gel size:

7 x 7 cm or 7 x 14 cm



•

Number of sample wells required:

6



•

Placement of well-former template:

first set of notches



•

Agarose gel concentration:

0.8%

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. A. Using Rubber dams:

•

Place a rubber dam on each end of the bed. Make sure the dam fits firmly in contact with the sides and bottom of the bed.

B. Taping with labeling or masking tape:

•

With 3/4 inch wide tape, extend the tape over the sides and bottom edge of the bed.



•

Fold the extended edges of the tape back onto the sides and bottom. Press contact points firmly to form a good seal.

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.

CASTING agarose Gels •

•

If you will be staining the gel after electrophoresis with InstaStain® Methylene Blue, you should be using Table A.1 or A.2 found in Appendix A. If you will be staining the gel after electrophoresis with InstaStain® Ethidium Bromide, you should be using Table A.3 or A.4 found in Appendix B.

3. Use a 250 ml flask or beaker to prepare the gel solution. 4. Use the appropriate Reference Table for agarose gel preparation provided by your instructor. Add the specified amount of agarose powder and buffer and swirl the mixture to disperse clumps of agarose powder. 5. With a marking pen, indicate the level of the solution volume on the outside of the flask.

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

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109 Agarose Gel Electrophoresis 6. Heat the mixture to dissolve the agarose powder.

At high altitudes, it is recommended to use a microwave oven to reach boiling temperatures.

A. Microwave method: • Cover the flask 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.



Important Note



B. Hot plate method: • Cover the flask 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. Check the solution carefully and continue heating until the final solution appears clear (like water). If "crystal" particles are visible, the agarose is not completely dissolved.

7. 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 5.

After the gel is cooled to 60°C: • If you are using rubber dams, go to step 9. • If you are using tape, continue with step 8. 8. Seal the interface of the gel bed and tape to prevent the agarose solution from leaking.

•



•

DO NOT POUR BOILING HOT AGAROSE INTO THE GEL BED.

60˚C

Hot agarose solution may irreversibly warp the bed.

Use a transfer pipet to deposit a small amount of 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 (60°C) agarose solution into the bed. 10. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes.

Preparing the gel for electrophoresis 11. After the gel is completely solidified, 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 the surface tension.

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

The Experiment



18

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

EDVO-Kit #

109 Agarose Gel Electrophoresis 12. Remove the comb by slowly pulling straight up. Do this carefully and evenly to prevent tearing the sample wells.

The Experiment

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 (found in Appendix A or B) on the instruction sheet provided by your instructor).

Reminder: 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

+

Red

15. Make sure that the gel is completely submerged under buffer before proceeding to loading the samples and conducting electrophoresis.

loadING the Samples Samples should be loaded into the wells of the gel in consecutive order. • For gels to be stained with InstaStain® Methylene blue, the amount of sample that should be loaded is 35-38 µl. • For gels to be stained with InstaStain® Ethidium Bromide, the amount of sample that should be loaded is 18-20 µl. Lane

1 2 3 4 5 6

Tube A B C D E F

DNA from crime scene cut with Enzyme 1 DNA from crime scene cut with Enzyme 2 DNA from Suspect 1 cut with Enzyme 1 DNA from Suspect 1 cut with Enzyme 2 DNA from Suspect 2 cut with Enzyme 1 DNA from Suspect 2 cut with Enzyme 2

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

EDVO-Kit #

19

109 Agarose Gel Electrophoresis Running the Gel 1. After the DNA samples are loaded, carefully snap the cover down onto the electrode terminals. Make sure that the negative and positive

color-coded indicators on the cover and apparatus chamber are properly oriented.

Electrophoresis can be completed in 15-20 minutes under optimal conditions. For Time and Voltage recommendations, refer to Table C (in Appendix A or B).

3. Set the power source at the required voltage and conduct electrophoresis for the length of time determined by your instructor. 4. Check to see that current is flowing properly - you should see bubbles forming on the two platinum electrodes. 5. After the electrophoresis is completed, turn off the power, unplug the power source, disconnect the leads and remove the cover. 6. 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. Various options are available for DNA staining. Your instructor will provide instructions for the DNA staining method you will be using.

InstaStain is a registered trademark of EDVOTEK, Inc. Patents Pending.

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

The Experiment

2. Insert the plug of the black wire into the black input of the power source (negative input). Insert the plug of the red wire into the red input of the power source (positive input).

20

EDVO-Kit #

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

109 Study Questions Answer the following study questions in your laboratory notebook or on a separate worksheet.

The Experiment

1. Define FLP’s and give their significance. 2. What is the most likely cause of Restriction Fragment Length Polymorphisms? 3. What are Variable Number of Tandem Repeats (VNTRs)? 4. Who are the only individuals possessing the same DNA fingerprints? 5. List the steps involved in DNA fingerprinting from extraction of DNA through the matching of a suspect to a crime scene sample. 6. What type of human cells can be utilized for this technique?

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, EDVOTEK, Inc., all rights reserved. EVT 001197AM The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

®

Material Safety Data Sheet

Date Prepared

10/05/06

Date Prepared

ACGIH TLV

Other Limits Recommended

No data

Flammable Limits

LEL UEL

No data available

Stable

Unstable

Will Not Occur

May Occur

Inhalation?

No data available

IARC Monographs?

Treat symptomatically and supportively

Normal solid waste disposal

Date Prepared

OSHA Regulation?

N.D.

UEL

None

Skin: Wash with soap and water

Section VII - Precautions for Safe Handling and Use

Yes

Other

Mechanical Gen. dilution ventilation

Work/Hygienic Practices

Splash proof goggles

None

Impervious clothing to prevent skin contact

Eye Protection

Special

Local Exhaust

Other Protective Clothing or Equipment

Protective Gloves

Ventilation

Yes

None None

May Occur Will Not Occur

None

None

Conditions to Avoid

Skin?

None

Conditions to Avoid

IARC Monographs?

Yes

No data Treat symptomatically and supportively

Ingestion? Yes

Protective Gloves

Ventilation

OSHA Regulation?

Mix material with combustible solvent and burn in a chemical incinerator equipped afterburner and scrubber

Mutagen

Yes Rubber

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

SCBA

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

Other Precautions

N.D.

UEL

Irritation to mucous membranes and upper respiratory tract Medical Conditions Generally Aggravated by Exposure

Signs and Symptoms of Exposure

Carcinogenicity: No data available NTP?

Work/Hygienic Practices

_Safety goggles

X Inhalation? Yes

Other Protective Clothing or Equipment

Eye Protection

Other

N.D.

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

Work/Hygienic Practices

Yes

Mechanical (General)

Local Exhaust

LEL

Carbon monoxide, Carbon dioxide, nitrogen oxides, hydrogen bromide gas Hazardous Polymerization

Other Protective Clothing or Equipment None

Protective Gloves

Ventilation

X Strong oxidizing agents Hazardous Decomposition or Byproducts

Incompatibility

Stable

Unstable

Section V - Reactivity Data

Emits toxic fumes

Unusual Fire and Explosion Hazards

Stability

Flammable Limits

No data

No data

No data

Wear protective clothing and SCBA to prevent contact with skin & eyes

Special Fire Fighting Procedures

Respiratory Protection (Specify Type)

Yes

No data

N.D. = No data

Evaporation Rate (Butyl Acetate = 1)

Melting Point

Specific Gravity (H 0 = 1) 2

% (Optional)

Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam

Extinguishing Media

Flash Point (Method Used)

Respiratory Protection (Specify Type)

None

Avoid eye and skin contact.

No data

No data

No data

Section IV - Physical/Chemical Characteristics

Respiratory Protection (Specify Type)

Other Precautions

Special

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.

Steps to be Taken in case Material is Released for Spilled

Yes

OSHA Regulation?

Ingestion?

Ingestion: If conscious, give large amounts of water

Eyes: Flush with water Inhalation: Move to fresh air

Emergency First Aid Procedures

Yes

IARC Monographs?

Skin?

None

Irritation to upper respiratory tract, skin, eyes

NTP?

Yes

Medical Conditions Generally Aggravated by Exposure

Signs and Symptoms of Exposure

Carcinogenicity: None identified

None

Inhalation?

Health Hazards (Acute and Chronic)

Route(s) of Entry:

X

Conditions to Avoid

Carbon monoxide, Carbon dioxide

Will Not Occur

May Occur

None

Conditions to Avoid

Strong oxidizing agents

X

None identified

Section VI - Health Hazard Data

Hazardous Polymerization

N.D.

Soluble

Section VIII - Control Measures

Chemical cartridge respirator with full facepiece.

Unstable Stable

LEL

Data not available

ACGIH TLV

Appearance and Odor Chemical bound to paper, no odor

Solubility in Water

Vapor Density (AIR = 1)

Vapor Pressure (mm Hg.)

No data

Boiling Point

No data

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

CAS# 139-33-3

Section III - Physical/Chemical Characteristics No data

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)

Appearance and Odor Clear, liquid, slight vinegar odor

Solubility in Water

No data

Melting Point

Specific Gravity (H 0 = 1) 2

OSHA PEL

10/05/06

Other Limits Recommended

Signature of Preparer (optional)

(2,7-Diamino-10-Ethyl-9-Phenylphenanthridinium Bromide)

Ethidium Bromide

Section VIII - Control Measures

None

None

Yes

ACGIH TLV

Section VIII - Control Measures

Other Precautions

Precautions to be Taken in Handling and Storing

Waste Disposal Method

Ingestion?

Sweep up and place in suitable container for disposal

Steps to be Taken in case Material is Released for Spilled

Section VII - Precautions for Safe Handling and Use

Emergency First Aid Procedures

Yes

None

N.D.

Ingestion: Large amounts may cause diarrhea

No data available

NTP?

Yes

Skin?

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)

No data

No data

Appreciable, (greater than 10%)

Vapor Pressure (mm Hg.) Vapor Density (AIR = 1)

Boiling Point

No data

Section III - Physical/Chemical Characteristics No data

N.D. = No data

OSHA PEL

This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard.

Melting Point

Specific Gravity (H 0 = 1) 2

White powder, no odor

Insoluble - cold

No data

No data

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.

OSHA PEL

(301) 251-5990

(301) 251-5990

Telephone Number for information

Emergency Telephone Number

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

InstaStain, Inc. P.O. Box 1232 West Bethesda, MD 20827

Manufacturer's Name

Section I

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.

Section II - Hazardous Ingredients/Identify Information % (Optional)

10/05/06

Other Limits Recommended

Signature of Preparer (optional)

(301) 251-5990

Telephone Number for information

(301) 251-5990

Emergency Telephone Number

InstaStain® Ethidium Bromide

IDENTITY (As Used on Label and List)

®

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

14676 Rothgeb Drive Rockville, MD 20850

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

EDVOTEK, Inc.

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.

EDVOTEK

Section II - Hazardous Ingredients/Identify Information % (Optional)

50x Electrophoresis Buffer Manufacturer's Name

Section I

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.

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

Signature of Preparer (optional)

®

IDENTITY (As Used on Label and List)

EDVOTEK

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.

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

Agarose

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.

Material Safety Data Sheets

®

Date Prepared

10/05/06 10/05/06

Soluble Blue liquid, no odor

No data

LEL

Will Not Occur

May Occur

Yes

IARC Monographs?

May cause skin or eye irritation

NTP?

Wear eye and skin protection and mop spill area. Rinse with water.

Steps to be Taken in case Material is Released for Spilled

Yes

Mechanical (General)

Work/Hygienic Practices

Avoid eye and skin contact

None required

Splash proof goggles

Other Eye Protection

None None

Special

Yes Yes

Local Exhaust

Other Protective Clothing or Equipment

Protective Gloves

Ventilation

Respiratory Protection (Specify Type)

Section VIII - Control Measures

None

Other Precautions

Avoid eye and skin contact.

Precautions to be Taken in Handling and Storing

Observe all federal, state, and local regulations.

Waste Disposal Method

OSHA Regulation?

Ingestion? Yes

Treat symptomatically and supportively. Rinse contacted area with copious amounts of water.

Section VII - Precautions for Safe Handling and Use

Emergency First Aid Procedures

Yes

Acute eye contact: May cause irritation. No data available for other routes.

Skin?

None

Conditions to Avoid

Medical Conditions Generally Aggravated by Exposure None reported

Signs and Symptoms of Exposure

No data available

Carcinogenicity:

X

Inhalation?

Health Hazards (Acute and Chronic)

Route(s) of Entry:

None

Conditions to Avoid

Sulfur oxides, and bromides

X

Unknown

Section VI - Health Hazard Data

Hazardous Polymerization

Hazardous Decomposition or Byproducts

None

Stable

Unstable

Section V - Reactivity Data

Incompatibility

UEL

No data No data

Use agents suitable for type of surrounding fire. Keep upwind, avoid breathing hazardous sulfur oxides and bromides. Wear SCBA.

Unusual Fire and Explosion Hazards

Stability

Flammable Limits

Dry chemical, carbon dioxide, water spray or foam

Special Fire Fighting Procedures

Extinguishing Media

Flash Point (Method Used)

Section IV - Physical/Chemical Characteristics

Appearance and Odor

Solubility in Water

No data

Evaporation Rate (Butyl Acetate = 1)

No data

Vapor Density (AIR = 1)

Soluble - cold

No data

No data

No data

Evaporation Rate (Butyl Acetate = 1)

Melting Point

No data available

No data

LEL

None

NTP?

Yes

IARC Monographs?

Eyes: May cause eye irritation

Skin?

None

Conditions to Avoid

Ingestion?

No data available

Treat symptomatically

OSHA Regulation?

Mix material with a combustible solvent and burn in chemical

Ventilate area and wash spill site

Rubber

Work/Hygienic Practices

Rubber boots

Mechanical (General)

Other

Special

Eye Protection

Required

MIOSH/OSHA approved, SCBA Local Exhaust

Other Protective Clothing or Equipment

Protective Gloves

Ventilation

Respiratory Protection (Specify Type)

Section VIII - Control Measures

None

Other Precautions

Keep tightly closed. Store in cool, dry place

Precautions to be Taken in Handling and Storing

Chem. safety goggles

incinerator equipped with afterburner and scrubber. Check local and state regulations.

Waste Disposal Method

Steps to be Taken in case Material is Released for Spilled

Section VII - Precautions for Safe Handling and Use

Emergency First Aid Procedures

Medical Conditions Generally Aggravated by Exposure No data available

Signs and Symptoms of Exposure

Yes Inhalation: Cyanosis Meets criteria for proposed OSHA medical records rule PEREAC 47.30420.82

Carcinogenicity:

Yes

X Inhalation?

Skin: May cause skin irritation

Health Hazards (Acute and Chronic)

Route(s) of Entry:

Conditions to Avoid

Toxic fumes of Carbon monoxide, Carbon dioxide, nitrogen oxides, sulfur oxides, hydrogen, chloride gas Will Not Occur

May Occur

Section VI - Health Hazard Data

Hazardous Polymerization

X

Emits toxid fumes under fire conditions

Strong oxidizing agents

Stable

Hazardous Decomposition or Byproducts

Incompatibility

Unstable

Section V - Reactivity Data

Unusual Fire and Explosion Hazards

Self contained breathing apparatus and protective clothing to prevent contact with skin and eyes

Special Fire Fighting Procedures

Stability

No data

UEL

No data

No data

No data

% (Optional)

Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam

Extinguishing Media

Flammable Limits

Section IV - Physical/Chemical Characteristics Flash Point (Method Used)

No data available

Other Limits Recommended

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

No data

No data

Melting Point

No data

Vapor Pressure (mm Hg.)

Specific Gravity (H 0 = 1) 2

Boiling Point

No data

Section III - Physical/Chemical Characteristics

ACGIH TLV

Section III - Physical/Chemical Characteristics

OSHA PEL

Methylene Blue 3.7 Bis (Dimethylamino) Phenothiazin 5 IUM Chloride CAS # 61-73-4

This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard.

ACGIH TLV

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

% (Optional)

Date Prepared Signature of Preparer (optional)

(301) 251-5990

Telephone Number for information

Emergency Telephone Number

Section II - Hazardous Ingredients/Identify Information

OSHA PEL

Other Limits Recommended

14676 Rothgeb Drive Rockville, MD 20850

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

EDVOTEK, Inc.

Manufacturer's Name

Section I (301) 251-5990

Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must

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.

InstaStain® Methylene Blue, Methylene Blue Plus™be marked to indicate that.

IDENTITY (As Used on Label and List)

®

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

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.

EDVOTEK

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

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.

Practice Gel Loading Solution

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.

Material Safety Data Sheets