DNA Fingerprinting II - Austin Community College District · DNA Fingerprinting II Usage of...
Transcript of DNA Fingerprinting II - Austin Community College District · DNA Fingerprinting II Usage of...
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225DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
Storage: See Page 3 for specifi c storage instructions
EXPERIMENT OBJECTIVE:
The objective of this simulated forensic analysis is to develop an understanding of the use of restriction
enzymes as applied to RFLP-based DNA fi ngerprinting.
Updated
Revised
and
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Table of Contents
Page
Experiment Components 3
Experiment Requirements 3
Background Information 5
Experiment Procedures
Experiment Overview and General Instructions 13
Restriction Enzyme Digestion 14
Electrophoresis
Agarose Gel Preparation 16
Conducting Electrophoresis 20
Staining and Visualization of DNA 22
Method 1: Staining with InstaStain® Methylene
Blue Cards
Method 2: One-Step Staining and Destaining
with InstaStain® Methylene Blue
Study Questions 25
Instructor's Guidelines 27
Notes to the Instructor 28
Pre-Lab Preparations
Restriction Enzyme Digestion 30
Electrophoresis 33
Quantity Prep for Agarose Gel Electrophoresis 34
Experiment Results and Analysis 35
Study Questions and Answers 36
Material Safety Data Sheets 37
All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor adminis-tered to or consumed by humans or animals.
THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experiment com-ponents are derived from human sources.
EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.
This experiment contains reagents and materials for six groups.
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Experiment Components
Contents Storage
A Crime scene DNA sample, -20°C freezer pre-cut with Restriction Enzyme 1B Crime scene DNA sample, -20°C freezer pre-cut with Restriction Enzyme 2
(Samples A and B are ready for electrophoresis)
C Suspect #1 DNA sample -20°C freezerD Suspect #2 DNA sample -20°C freezerE Standard DNA Fragments -20°C freezerF Enzyme Reaction Buffer Room tempG Dryzymes™ Restriction Enzyme 1 (Eco RI) Room tempH Dryzymes™ Restriction Enzyme 2 (Hind III) Room tempI Reconstitution buffer -20°C freezerJ Enzyme Grade water -20°C freezer
• 10x Gel Loading Solution• UltraSpec-Agarose™ powder• Concentrated electrophoresis buffer• InstaStain® Methylene Blue • 1 ml pipet• 100 ml plastic graduated cylinder • Microtipped Transfer Pipets
• Horizontal gel electrophoresis apparatus• D.C. power supply• Automatic micropipets with tips• Water bath (45°C)• Balance• Hot plate, Bunsen burner or microwave oven• DNA visualization system (white light)• Small plastic trays or large weigh boats (for gel destaining)
• Safety goggles and disposable laboratory gloves• Pipet pumps• 20 ml and 250 ml beakers or fl asks• Hot gloves• Marking pens• Distilled or deionized water• Ice
Requirements
This experiment contains reagents for six groups.
DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
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5DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
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Bac
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und Info
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DNA Fingerprinting
Figure 1: Examples of restriction enzymes and the organism of origin.
When fi rst introduced, DNA fi ngerprinting (also called DNA profi le analysis or DNA typing) involved the electrophoretic analysis of DNA fragment sizes generated by restriction enzymes. In contrast to more conventional methodologies, such as blood typing, which excludes suspects, traditional DNA fi ngerprinting provides accurate, unambiguous identifi cation of the source of DNA samples.
Variations in DNA sequences between individuals as determined by dif-ferences in restriction enzyme cleavage patterns are known as restriction fragment length polymorphisms (RFLPs). RFLPs are a manifestation of the unique molecular genetic profi le, or “fi ngerprint”, of an individual’s DNA.
RESTRICTION ENZYMES
Bgl I Bactillus globigii
Bam HI Bacillus amyloliquefaciens H
Eco RI Escherichia coli RY13
Eco RII Escherichia coli R 245
Hae III Haemophilus aegyptius
Hind III Haemophilus influenzae R4
Restriction Enzyme Organism
Restriction enzymes are endonucleases that catalyze cleavage of phos-phate bonds. They require Mg+2 for activity and generate a 5 prime (5') phosphate and a 3 prime (3') hydroxyl group at the point of cleav-age. The distinguishing feature of restriction enzymes compared to other endonucleases is that they only cut at very specifi c sequences of bases. Restriction enzymes are produced by many different species of bacteria (including blue-green algae). Over 3,000 restriction enzymes have been discovered and catalogued.
Restriction enzymes are named according to the organism from which they are isolated. The fi rst letter of the genus followed by the fi rst two letters of the species (Fig. 1). Only certain strains or sub-strains of a particular species may produce restriction enzymes. The type of strain or substrain sometimes follows the species designation in the name. Finally, a Roman numeral is used to designate one out of several restriction enzymes produced by the same organism.
A restriction enzyme requires a specifi c double-stranded recognition sequence of nu-cleotide bases to cut DNA. Recognition sites are generally 4 to 8 base pairs in length and cleavage occurs within or near that site. Rec-ognition sites are frequently symmetrical, i.e., both DNA strands in the site have the same base sequence when read 5' to 3' and the cleavage positions are indicated by arrows. Such sequences are called palindromes.
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225DNA Fingerprinting II
Usage of Restriction Enzymes in DNA Fingerprinting Analysis
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Consider the recognition site and cleavage pattern of Eco RI as an ex-ample. Eco RI causes staggered cleavage of its site. The resulting ends of the DNA fragments are called “sticky” or “cohesive” ends.
↓ 5'-GAATTC-3' 5'-G AATTC-3' 3'-CTTAAG-5' 3'-CTTAA G-5' ↑
In DNA forensics laboratories, the two most commonly used restriction enzymes were Hae III and Hinf I, which are 4-base and 5-base cutting enzymes.
Hae III ↓ 5'-GGCC-3' 3'-CCGG-5' ↑
Hinf I ↓ 5'-GANTC-3' 3'-CTNAG-5' ↑
The size of DNA fragments generated depends on distances between the recognition sites. In general, the longer the DNA molecule, the greater the probability that a given recognition site will occur. The DNA of an av-erage human chromosome is very large, containing over 100 million 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.
No two individuals have exactly the same pattern of restriction enzyme recognition sites. There are several reasons for this fact that exists in a population. Alleles are alternate forms of a gene that result in alternative expressions of genetic traits that can be dominant or recessive. Chromo-somes occur in matching pairs, one of maternal and the other of paternal origin. The two copies of a gene at a given chromosomal locus represent a composite of parental genes and constitute an individual’s unique genotype. It follows that alleles have differences in their base sequences which consequently creates differences in the distribution and frequen-cies 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 restriction endo-nuclease palindromic site. The example in Figure 2 shows how a silent mutation can eliminate a recognition site but leave a protein product unchanged.
DNA Fingerprinting
7DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
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Individual variations in the distances between recogni-tion sites in chromo-somal DNA are often caused by interven-ing repetitive base sequences. Rep-etitious sequences constitute a large fraction of the mam-malian genome and have no known genetic function. These sequences can occur between
genes or are adjacent to them. They are also found within introns. Ten to fi fteen percent of mammalian DNA consists of sets of repeated, short sequences of bases that are tandemly arranged in arrays. The length of these arrays (the amount of repeated sets) varies between individuals at different chromosomal loci.
TGTTTA|TGTTTA|TGTTTA|.........variable number
When these arrays are fl anked by recognition sites, the length of the repeat will determine the size of the restriction enzyme fragment gener-ated. Several types of short, repetitive sequences have been cloned and sequenced.
AGAROSE GEL ELECTROPHORESIS
Agarose gel electrophoresis is used to analyze DNA fragments generated by restriction enzymes. Agarose gels consist of microscopic pores that act as a molecular sieve. DNA fragments 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 elec-trophoresis. DNA fragments are separated by the gel according to their size, charge and shape. DNA fragments are linear and the ratio of mass to charge is the same. Therefore, only the size of the fragment affects the mobility. The smaller the fragment the faster it migrates. After electropho-resis, DNA can be visualized by staining.
Restriction enzyme cleavage of relatively small DNA molecules, such as plasmids and viral DNAs, usually results in discrete banding patterns of DNA fragments after electrophoresis. However, cleavage of large and complex DNA, such as human chromosomal DNA, generates many differ-ently sized fragments that the resolving capacity of the gel is exceeded.
Eco RI site
5' ACG AAT TCC 3' Coding DNA
2H N Thr - Asn - Ser COOH Protein
5' ACG AAC TCC 3' Codon changed by mutation
Thr - Asn - Ser COOH Protein
*
2H N
DNA Fingerprinting
Figure 2: Silent mutation (T C) changes the Eco RI site.
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Usage of Restriction Enzymes in DNA Fingerprinting Analysis
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Consequently, the cleaved chromosomal DNA is visualized as a smear after staining and has no obvious banding patterns.
SOUTHERN BLOT ANALYSIS
RFLP analysis of genomic DNA is facilitated by Southern blot analysis. After electrophoresis, DNA fragments in the gel are denatured by soaking in an alkali solu-tion. This causes double-stranded 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 transfer-ring (blotting) them to a sheet of nitrocellulose or nylon membrane (Figure 3). This is done by plac-ing the membrane on the gel after electropho-resis and transferring DNA
DNA Fingerprinting
fragments to the membrane by capillary action or electrotransfer. DNA, which is not visible, becomes permanently adsorbed to the membrane, that can then be manipulated easier than gels.
Analysis of the blotted DNA is done by hybridization with a labeled oligo-nucleotide DNA probe. The probe is a DNA fragment that contains base sequences that are complementary to the variable arrays of tandemly repeated sequences found in the human chromosomes. Probes can be labeled with reporter molecules that are used for detection. A solution containing the single-stranded probe is incubated with the membrane containing the blotted, single-stranded (denatured) DNA fragments. Under the proper conditions, the probe will only base pair (hybridize) to those fragments containing the complementary sequences. The mem-brane is then washed to remove excess probe. Only DNA fragments that
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Evidence
Suspect's Blood
1. Collection of DNA2. Extraction of DNA3. DNA cut into fragments by restriction enzymes4. DNA fragments separated by agarose gel electrophoresis5. DNA denatured into single strands6. DNA blotted on a nylon membrane (Southern Blot)7. Nylon membrane soaked with probes that bind to target DNA fragments and detected.8. Computer analysis
6
Southern Blot
4
Figure 3: DNA Fingerprinting using RFLP and Southern blot analysis.
9DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
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are hybridized to the probe will reveal their positions on the membrane. If the probes are isotopically labeled, the hybridized fragments will appear as discrete bands (fi ngerprint) on the fi lm and are in the same relative po-sitions as they were in the agarose gel after electrophoresis. Only specifi c DNA fragments of the hundreds of thousands of fragments present, will hybridize with the probe because of the selective nature of the hybridiza-tion process.
In forensic analysis, DNA samples can be extracted and purifi ed from specimens of skin, blood stains, semen, or hair roots collected at the crime scene. RFLP analyses performed on these samples is then compared to those performed on samples obtained from the suspect. If RFLP patterns match, it is beyond reasonable doubt that the suspect (or biological ma-terial from the suspect, such as blood) was at the crime scene. In forensic DNA fi ngerprinting, different sets of probes hybridized to different types
DNA Fingerprinting
of repetitious sequences are used in DNA profi le analysis in order to satisfy certain statistical criteria for positive identifi cation.
DNA FINGERPRINTING USING
POLYMERASE CHAIN REACTION (PCR)
RFLP-based DNA fi ngerprinting analysis has been overtaken by the Polymerase Chain Reaction (PCR) because of two important advantages. The fi rst is the sensitivity of PCR, which allows for DNA fi ngerprinting identifi cation using much smaller amounts of DNA since PCR amplifi es DNA. A second advantage is the speed of PCR analysis, which allows critical questions to be answered more quickly as compared to Southern Blot analysis.
PCR amplifi cation requires the use of a thermostable DNA polymerase, such as Taq polymerase. Purifi ed from a bacte-rium 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
2
1
Evidence
Suspect's Blood
1. Collection of DNA2. Extraction of DNA3. DNA Amplification (PCR)4. DNA fragments separated by agarose gel electrophoresis5. Analysis
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3 5'3'3'5'
5'3'
5'
5'
5'3'3'5'
5'
5'5' 3'
5'3'
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5'3'
5'
5' 3'
5'3'3'5'
5'
5'
5'
5'3'
5' 3'
5' 3'
5' 3'
Figure 4: DNA Fingerprinting using PCR
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225DNA Fingerprinting II
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3' 5'
3' 5'
5' 3'
5' 3'
5'
5' 3' 3' 5'
5' 3'
5' 5'
Denature 94°C
5'
Extension72°C
3' 5'
Separation of 2 DNA strands
=
Primer 1 =
Primer 2 =
5' 3' 5'
Anneal 2 primers
45°C
3' 5' 5'
5' 5'
3' 5' 5'
5'
5' 3'
5'
5' 5'
5' 3'
5' 3'
5' 3'
5' 3'
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5' 3'
5'
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Cyc
le 1
C
ycle
2
Cyc
le 3
Target Sequence
5' 3'
5' 3'
5' 3'
Figure 5: The Polymerase Chain Reaction
DNA Fingerprinting
11DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
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as "primers". These components, together with the DNA to be amplifi ed, 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 ampli-fi ed, known as the "target".
The PCR reaction mixture (which contains the DNA polymerase, buffer, deoxynucleotides, primers, and template) is subjected to sequential heat-ing/cooling cycles at three different temperatures (Figure 5).
• In the fi rst step, the template 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 sepa-rated 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 complete the synthesis of the new complimentary strands.
DNA fi ngerprinting analysis has become increasingly signifi cant in court cases involving murder, rape, physical battery, and other types of crimes. Jurors are often asked to determine the validity of DNA evidence, result-ing in both acquittals and convictions of suspected criminals. To ensure greater accuracy, scientists have incorporated standardization proce-dures in DNA analysis. Standard DNA Fragments are used to determine the exact size of individual DNA fragments in a DNA fi ngerprint. It is generally accepted that DNA fi ngerprints are identical only in the case of identical twins.
In this experiment, emphasis is placed on concepts related to RFLP analy-sis. The experiment activities will focus on the identifi cation of DNA by analyzing restriction fragmentation patterns separated by agarose gel electrophoresis.
THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA.
DNA Fingerprinting
DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
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Experiment Notes
13DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
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The Exp
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EXPERIMENT OBJECTIVE:
The objective of this simulated forensic analysis is to develop an under-standing of the use of restriction enzymes as applied to RFLP-based DNA fi ngerprinting.
LABORATORY SAFETY
1. Gloves and goggles should be worn routinely as good laboratory practice.
2. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents.
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4. Exercise caution when using any electrical equipment in the laboratory.
5. Always wash hands thoroughly with soap and water after handling reagents or bio-logical 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 refl ects 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 ex-
periment were repeated. • Write a hypothesis that would refl ect this change.
Experiment Overview and General Instructions
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory 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 © 1996, 1997, 1998, 2000, 2001, 2006, EDVOTEK, Inc., all rights reserved EVT 002127K
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225DNA Fingerprinting II
Usage of Restriction Enzymes in DNA Fingerprinting Analysis
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Reaction tube 1
Reaction tube 2
Suspect 1 DNA
25 µl 25 µl
Enzyme 1 Enzyme 2
Add 15 µl to reaction tube 2
Add 15 µl to reaction tube 1 40 µl
45 µl
10 µl
Reaction Buffer
10 µl
40 µl
45 µl
Incubate at37°C for
30 - 60 minutes
Add 5 µlGel Loading
Solution
Samplesare ready for
electrophoresis
Add 10 µl to each reaction
tube
Add 15 µl to both reaction
tubes
Quick Reference:
Dispensed Components Tube Label
Crime scene DNA 1 CS 1Crime scene DNA 2 CS 2Suspect 1 DNA DNA 1Suspect 2 DNA DNA 2Standard DNA fragments MarkersEnzyme Reaction Buffer Rxn Buffer Diluted Enzyme 1 Enzyme 1Diluted Enzyme 2 Enzyme 2
In this experiment, the DNA from two suspects are each digested with two restriction enzymes in sepa-rate reactions and compared to crime scene samples after agarose gel electrophoresis. This fl ow chart outlines the procedure used for the restriction enzyme digestion of DNA obtained from Suspect 1. The DNA from Suspect 2 is digested in the same manner, using reaction tubes 3 and 4 (not shown).
Crime Scene Investigation - Restriction Enzyme Digestion
To avoid cross-contamination, use a FRESH micropipet tip for each transfer of DNA and enzyme to the restriction enzyme reaction.
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Restriction Enzyme Digestion
1. Label microtest tubes 1 through 4 for four restriction enzyme digestion reactions. Put your initials or group number on the tubes.
2. Tap all the tubes (see Quick Reference at left) on the lab bench to
collect all the contents at the bottom of the tube. 3. Use an automatic micropipet to dispense 10 µl of Enzyme Reaction
Buffer (Rxn Buffer) to each of four reaction tubes labeled 1 through 4.
4. Add DNA and enzyme to the reaction tubes as summarized in Chart 1. Use a FRESH micropipet tip for each transfer of DNA and enzyme.
* 10x Gel loading solution has already been added to the crime scene samples.
Chart 1: Summary of Restriction Enzyme Digestion Reactions
Crime Scene Samples
Suspect 1
Suspect 2
Reaction Reaction Enzyme 1 Enzyme 2 Final Tube Buffer DNA 1 (µl) DNA 2 (µl) (µl) (µl) Volume (µl)
OPTIONAL STOPPING POINT
After addition of 10x gel loading solution to stop the reaction, samples are ready for electrophoresis. Samples may be stored in the refrigerator for electrophoresis.
The enzymes used in this experiment are stored and shipped in Dryzyme™ form (lyophilized). A buffer has been added to reconstitute the enzyme to liquid form.
5. Cap the reaction tubes and tap gently to mix. Then tap each tube on the lab bench to collect contents at the bottom.
6. Incubate reaction tubes in a 37°C waterbath for 30 minutes (or 60 minutes if time allows).
After the 30 or 60 minute incubation is completed:
7. Add 5 µl of 10x gel loading solution to reaction tubes 1 - 4 to stop the reactions. Cap and mix by tapping.
Crime Scene DNA, cut with enzyme 1 ready for electrophoresis X -- 45 *
Crime Scene DNA, cut with enzyme 2 ready for electrophoresis -- X 45 *
1 10 15 -- 15 -- 40
2 10 15 -- -- 15 40
3 10 -- 15 15 -- 40
4 10 -- 15 -- 15 40
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AGAROSE GEL REQUIREMENTS FOR THIS EXPERIMENT
• Recommended gel size: 7 x 7 cm or 7 x 14 cm
• Number of sample wells required: 7 - 8 • Well-former template (comb):
For EDVOTEK units: 7 x 14 cm gel - two standard 6-well combs in the fi rst and middle set of notches 7 x 7 cm gel - one 8-well comb in the fi rst 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 rubber dam fi ts fi rmly 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 fi rmly to form a good seal.
2. Placement of well-former template (comb): For EDVOTEK units: 7 x 14 cm gel - two standard 6-well combs in the fi rst and middle set of notches 7 x 7 cm gel - one 8-well comb in
the fi rst set of notches
Electrophoresis - Agarose Gel Preparation
17DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
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Electrophoresis - Agarose Gel Preparation
CASTING THE AGAROSE GEL(S)
3. Use a 250 ml fl ask or beaker to prepare the gel solution.
Amt ofAgarose
(g)
ConcentratedBuffer (50x)
(ml)
Size of Gel(cm)
DistilledWater(ml)
TotalVolume
(ml)
7 x 7
7 x 14
0.23
0.46
0.6
1.2
29.4
58.8
30
60
+ =+
Individual 0.8%* UltraSpec-Agarose™ Gel
DNA Staining with InstaStain® MetBlue
Table
A.1
*0.77% UltraSpec-Agarose™ gel percentage rounded up to 0.8%
Amt ofAgarose
(g)
DilutedBuffer (1x)
(ml)
Size of Gel(cm)
7 x 7
7 x 14
0.23
0.46
30
60
+
Table
A.2 Individual 0.8%* UltraSpec-Agarose™ Gel
DNA Staining with InstaStain® MetBlue
*0.77% UltraSpec-Agarose™ gel percentage rounded up to 0.8%
If preparing the gel with concentrated (50x) buffer, use Table A.1.
IMPORTANT
Check with your instructor regarding the concentration of the buffer you are using to prepare your gel. Use the appropriate table (A.1 or A. 2) below.
If preparing the gel with diluted (1x) buffer, use Table A.2.
4. 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 fl ask.
Diluted buffer is onevolume of concentrated buffer to every 49 volumes of distilled or deionized water. See Table B.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory 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 © 1996, 1997, 1998, 2000, 2001, 2006, EDVOTEK, Inc., all rights reserved EVT 002127K
18
225DNA Fingerprinting II
Usage of Restriction Enzymes in DNA Fingerprinting Analysis
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
EDVO-Kit #Th
e E
xpe
rime
nt
6. Heat the mixture to dissolve the agarose powder. The fi nal solution should appear clear (like water) without any undissolved particles.
A. Microwave method:
• Cover the fl ask with plastic wrap to minimize evaporation.
• Heat the mixture on High for 1 minute.
• Swirl the mixture and heat on High in bursts of 25 seconds until all the agarose is completely dissolved.
B. Hot plate method:
• Cover the fl ask with aluminum foil to prevent excess evapora-tion.
• Heat the mixture to boiling over a burner with occasional swirling. Boil until all the agarose is completely dissolved.
Check the solution carefully. If you see "crystal" particles, the agarose is not completely dissolved.
Electrophoresis - Agarose Gel Preparation
At high altitudes, it is recommended to use a microwave oven to reach boiling temperatures.
7. Cool the agarose solution to 60°C with careful swirling to promote even dissipa-tion of heat. If detectable evapora-tion has occurred, add distilled water to bring the solution up to the original volume marked on the fl ask in step 6.
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.
• 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. Pour the cooled agarose solution into the bed. Make sure the bed is on a level surface.
10. Allow the gel to completely solidify. It will become fi rm and cool to the touch after approximately 20 minutes.
DO NOT POUR BOILING HOT AGAROSE INTO THE GEL BED.
Hot agarose solution may irreversibly warp the bed.
60˚C
19DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
225EDVO-Kit #
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory 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 © 1996, 1997, 1998, 2000, 2001, 2006, EDVOTEK, Inc., all rights reserved EVT 002127K
The Exp
erim
ent
Electrophoresis - Agarose Gel Preparation
PREPARING THE GEL FOR ELECTROPHORESIS
11. After the gel is completely solidifi ed, 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 remov-ing the rubber dams. A thin plastic knife, spatula or pipet tip can be inserted between the gel and the dams to break possible surface ten-sion.
12. Remove the comb by slowly pull-ing straight up. Do this carefully and evenly to prevent tearing the sample wells.
13. Place the gel (on its bed) into the electrophoresis chamber, properly oriented, centered and level on the platform.
14. Fill the electrophoresis apparatus chamber with the appropriate amount of diluted (1x) electrophoresis buffer.
15. Make sure that the gel is completely submerged under buffer before proceeding to loading the samples and conducting electrophoresis.
For DNA analysis, the recom-mended electrophoresis buf-fer is Tris-acetate-EDTA, pH 7.8. The formula for diluting EDVOTEK (50x) concen-trated buffer is one volume of buffer concentrate to every 49 volumes of distilled or deionized water. Prepare buffer as required for your electrophoresis unit. 50x Conc.
Buffer (ml)Distilled
Water (ml)
6
8
10
20
294
392
490
980
+EDVOTEKModel #
Total Volume Required (ml)
Electrophoresis (Chamber) Buffer
M6+
M12
M36 (blue)
M36 (clear)
300
400
500
1000
Dilution
Table
B
IMPORTANT: Check with your instructor to determine if the buffer has previously been diluted. Pour the appropriate amount of 1x buffer into the electrophoresis chamber according to Table B below.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory 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 © 1996, 1997, 1998, 2000, 2001, 2006, EDVOTEK, Inc., all rights reserved EVT 002127K
20
225DNA Fingerprinting II
Usage of Restriction Enzymes in DNA Fingerprinting Analysis
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
EDVO-Kit #Th
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xpe
rime
nt
LOAD THE SAMPLES
1. Optional Step: Heat the samples, including the Stan-
dard DNA fragments for two minutes at 65°C. Allow the samples to cool for a few minutes.
2. Load 40 µl of each of the DNA samples in the following manner (7 x 14 cm gel):
Electrophoresis - Conducting Electrophoresis
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.
RUNNING THE GEL
1. After the DNA samples are loaded, carefully snap the cover down onto the electrode termi-nals.
Make sure that the negative and positive color-coded indicators on the cover and apparatus chamber are properly oriented.
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).
First Row Lane Tube
1 Markers Standard DNA Fragments 2 CS 1 DNA from crime scene cut with Enzyme 1 3 CS 2 DNA from crime scene cut with Enzyme 2 4 1 DNA from Suspect 1 cut with Enzyme 1 5 2 DNA from Suspect 1 cut with Enzyme 2
Second Row Lane Tube
1 Markers Standard DNA Fragments 2 3 DNA from Suspect 2 cut with Enzyme 1 3 4 DNA from Suspect 2 cut with Enzyme 2
Electrophoresis
M12
_Black
+RedSample
wells
21DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
225EDVO-Kit #
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory 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 © 1996, 1997, 1998, 2000, 2001, 2006, EDVOTEK, Inc., all rights reserved EVT 002127K
The Exp
erim
ent
Electrophoresis - Conducting Electrophoresis
4. Check to see that current is fl owing 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.
ABOUT DNA GEL STAINING After electrophoresis, the agarose gels require staining in order to visualize the separated DNA samples. This experiment features a proprietary stain called InstaStain® Methylene Blue. Two options are provided for using the InstaStain® Methylene Blue cards. Check with your instructor regarding which staining method you should use.
Method 1: One-step Staining and Destaining with InstaStain® MetBlue
Method 2: Staining with InstaStain® Methylene Blue
3. Set the power source at the required voltage and conduct electro-phoresis for the length of time determined by your instructor. General guidelines are presented in Table C.
Time and VoltageRecommendations
Minimum / Maximum
Volts
150
125
70
50
15 / 20 min
20 / 30 min
35 / 45 min
50 / 80 min
Table
CEDVOTEK Electrophoresis ModelM6+ M12 & M36
Minimum / Maximum
25 / 35 min
35 / 45 min
60 / 90 min
95 / 130 min
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory 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 © 1996, 1997, 1998, 2000, 2001, 2006, EDVOTEK, Inc., all rights reserved EVT 002127K
22
225DNA Fingerprinting II
Usage of Restriction Enzymes in DNA Fingerprinting Analysis
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
EDVO-Kit #Th
e E
xpe
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nt
InstaStain™
InstaStain™
Electrophoresis - Staining and Visualization of DNA
METHOD 1: ONE-STEP STAINING AND DESTAINING WITH INSTASTAIN® METHYLENE BLUE
Agarose gels can be stained and destained in one easy step with In-staStain™ Methylene Blue cards. This one-step method can be complet-ed in approximately 3 hours, or can be left overnight.
Wear gloves and safety goggles
Do not stain gel(s) in the electrophoresis apparatus.
One Step Stain and Destain
1. Remove the 7 x 14 cm agarose gel from its bed and completely sub-merse the gel in a small, clean tray containing 150 ml of distilled or de-ionized water, or used electrophore-sis buffer. The agarose gel should be completely covered with liquid.
Examples of small trays include large weigh boats, or small plastic food con-tainers
2. Gently fl oat two 7 x 7 cm cards of InstaStain® MetBlue with the stain side (blue) facing the liquid.
3. Let the gel soak undisturbed in the liquid for approximately 3 hours. The gel can be left in the liquid overnight (cover with plastic wrap to prevent evaporation).
4. After staining and destaining, the gel is ready for visualization and photography.
STORAGE AND DISPOSAL OF INSTASTAIN® METHYLENE BLUE CARDS AND GELS
• Stained gels may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid.
DO NOT FREEZE AGAROSE GELS!
• Used InstaStain® cards and destained gels can be discarded in solid waste disposal.
• Destaining solutions can be disposed down the drain.
23DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
225EDVO-Kit #
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory 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 © 1996, 1997, 1998, 2000, 2001, 2006, EDVOTEK, Inc., all rights reserved EVT 002127K
The Exp
erim
ent
METHOD 2: STAINING WITH INSTASTAIN® METHYLENE BLUE CARDS
1. After electrophoresis, place the agarose gel on a fl at surface covered with plastic wrap.
2. Wearing gloves, place the blue dye side of two InstaStain® Methylene Blue cards on the gel.
3. Firmly run your fi ngers several times over the entire surface of the InstaStain® cards to establish good contact between the InstaStain® cards and the gel.
4. To ensure continuous contact between the gel and the InstaStain® cards, place a gel casting tray and weight, such as a small empty beaker, on top of the InstaStain® cards.
5. Allow the InstaStain® Methylene Blue to sit on the gel for 5 to 10 minutes.
6. After staining, remove the InstaStain® cards.
If the color of the gel appears very light, wet the gel surface with buffer or distilled water and place the InstaStain® cards back on the gel for an additional 5 minutes.
Destaining and Visualization of DNA
7. Transfer the gel to a large weigh boat or small plastic container.
8. Destain with distilled water.*
• Add approximately 150 ml of distilled water to cover the gel.
9. Repeat destaining by changing the distilled water as needed.
Electrophoresis - Staining and Visualization of DNA
Wear gloves and safety goggles
InstaStain is a registered trademark of EDVOTEK, Inc. Patents Pending.
1
2
3
4
5
6
Place gel on a flatsurface covered with plastic wrap.
Place the InstaStain®card on the gel.
Place a small weightfor approx. 5 minutes.
Transfer to a smalltray for destaining.
Destain with 37°Cdistilled water.
Press firmly.
-----
-----
InstaStain™
InstaStain™
InstaStain™
InstaStain™
InstaStain™
InstaStain™
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory 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 © 1996, 1997, 1998, 2000, 2001, 2006, EDVOTEK, Inc., all rights reserved EVT 002127K
24
225DNA Fingerprinting II
Usage of Restriction Enzymes in DNA Fingerprinting Analysis
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
EDVO-Kit #Th
e E
xpe
rime
nt
Electrophoresis - Staining and Visualization of DNA
The larger DNA bands will initially be visible as dark blue bands against a lighter blue background. When the gel is completely destained, the larger DNA bands will become sharper and the smaller bands will be visible. With additional destaining, the entire background will become uniformly light blue.
10. Carefully remove the gel from the destain solution and examine the gel on a Visible Light Gel Visualization System. To optimize visibility, use the amber fi lter provided with EDVOTEK equipment.
11. If the gel is too light and bands are diffi cult to see, repeat the staining and destaining procedures.
* Destaining Notes
• Warmed distilled water at 37°C will accelerate destaining. Destaining will take longer with room temperature water.
• DO NOT EXCEED 37°C ! Warmer temperatures will soften the gel and may cause it to break.
• The volume of distilled water for destaining depends upon the size of the tray. Use the smallest tray available that will accommodate the gel. The gel should be completely submerged during destaining.
• Do not exceed 3 changes of water for destaining. Excessive destaining will cause the bands to be very light.
STORAGE AND DISPOSAL OF INSTASTAIN® METHYLENE BLUE CARDS AND GELS
• Stained gels may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid.
DO NOT FREEZE AGAROSE GELS!
• Used InstaStain® cards and destained gels can be discarded in solid waste disposal.
• Destaining solutions can be disposed down the drain.
25DNA Fingerprinting IIUsage of Restriction Enzymes in DNA Fingerprinting Analysis
225EDVO-Kit #
The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory 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 © 1996, 1997, 1998, 2000, 2001, 2006, EDVOTEK, Inc., all rights reserved EVT 002127K
The Exp
erim
ent
Answer the following study questions in your laboratory notebook or on a separate worksheet.
1. Which suspect’s DNA matches that found at the crime scene? Does this automatically mean that the suspect is guilty?
2. What possible experimental problems could occur to invalidate the results?
3. If only Restriction Enzyme 1 was used, would the interpretation be the same?
Study Questions
37
225EDVO-Kit #Material Safety Data Sheets
Full size (8.5 x 11”) pdf copy of MSDS available at www.edvotek.com or by request.
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
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0.12
00 S
tan
dar
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ust
be
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spec
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req
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ts.
IDEN
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(A
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sed
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Lab
el a
nd
Lis
t)N
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: B
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k sp
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are
no
t p
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If a
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VI -
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)
Car
cin
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NTP
?O
SHA
Reg
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IAR
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Sig
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Gen
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by
Exp
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Emer
gen
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Aid
Pro
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s
Sect
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VII
- Pr
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ns
for
Safe
Han
dlin
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Use
Step
s to
be
Take
n in
cas
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Was
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Prec
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Res
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Spec
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Pro
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Equ
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Pro
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No
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No
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X
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Y
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Yes
Inh
alat
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: N
o d
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avai
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In
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tio
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Larg
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may
cau
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No
dat
a av
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No
dat
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Trea
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tom
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up
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Swee
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pla
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su
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dis
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No
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No
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Ch
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spir
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fu
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cep
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.
ED
VO
TE
K®
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
50x
Elec
tro
ph
ore
sis
Bu
ffer
This
pro
du
ct c
on
tain
s n
o h
azar
do
us
mat
eria
ls a
s d
efin
ed b
y th
e O
SHA
Haz
ard
Co
mm
un
icat
ion
Sta
nd
ard
.
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
Ap
pre
ciab
le, (
gre
ater
th
an 1
0%)
Cle
ar, l
iqu
id, s
ligh
t vi
neg
ar o
do
r
No
dat
a
N.D
. = N
o d
ata N.D
.
N.D
.
Use
ext
ing
uis
hin
g m
edia
ap
pro
pri
ate
for
surr
ou
nd
ing
fir
e.
Wea
r p
rote
ctiv
e eq
uip
men
t an
d S
CB
A w
ith
fu
ll fa
cep
iece
op
erat
ed in
po
siti
ve p
ress
ure
mo
de.
No
ne
iden
tifi
ed
10/0
5/06
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
X
N
on
e
Stro
ng
oxi
diz
ing
ag
ents
Car
bo
n m
on
oxi
de,
Car
bo
n d
ioxi
de
X
N
on
e
Yes
Y
es
Y
es
No
ne
No
ne
iden
tifi
ed
Irri
tati
on
to
up
per
res
pir
ato
ry t
ract
, ski
n, e
yes
No
ne
Ing
esti
on
: If
co
nsc
iou
s, g
ive
larg
e am
ou
nts
of
wat
er
Eyes
: Fl
ush
wit
h w
ater
In
hal
atio
n:
Mo
ve t
o f
resh
air
Sk
in:
Was
h w
ith
so
ap a
nd
wat
er
Wea
r su
itab
le p
rote
ctiv
e cl
oth
ing
. M
op
up
sp
ill
and
rin
se w
ith
wat
er, o
r co
llect
in a
bso
rpti
ve m
ater
ial a
nd
dis
po
se o
f th
e ab
sorp
tive
mat
eria
l.
Dis
po
se in
acc
ord
ance
wit
h a
ll ap
plic
able
fed
eral
, sta
te, a
nd
loca
l en
viro
men
tal r
egu
lati
on
s.
Avo
id e
ye a
nd
ski
n c
on
tact
.
No
ne
Yes
N
on
e
Yes
N
on
e
Yes
_Saf
ety
go
gg
les
No
ne
No
ne
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
used
to
com
ply
with
OSH
A's
Haz
ard
Com
mun
icat
ion
Stan
dard
. 29
CFR
191
0.12
00 S
tand
ard
mus
t be
con
sulte
d fo
rsp
ecifi
c re
quir
emen
ts.
IDEN
TIT
Y (
As
Use
d on
Lab
el a
nd L
ist)
Not
e: B
lank
spa
ces
are
not
perm
itted
. If
any
item
is n
ot
appl
icab
le, o
r no
info
rmat
ion
is a
vaila
ble,
the
spa
ce m
ust
be m
arke
d to
indi
cate
tha
t.
Sect
ion
IM
anuf
actu
rer'
s N
ame
Sect
ion
II -
Haz
ardo
us In
gred
ient
s/Id
entif
y In
form
atio
n
Emer
genc
y Te
leph
one
Num
ber
Tele
phon
e N
umbe
r fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sign
atur
e of
Pre
pare
r (o
ptio
nal)
Add
ress
(N
umbe
r, St
reet
, City
, Sta
te,
Zip
Cod
e)
EDVO
TEK
, Inc
.
1467
6 R
othg
eb D
rive
Roc
kvill
e, M
D 2
0850
Haz
ardo
us C
ompo
nent
s [S
peci
fic
Che
mic
al Id
entit
y; C
omm
on N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imits
R
ecom
men
ded
% (
Opt
iona
l)
(301
) 25
1-59
90
(301
) 25
1-59
90
Boili
ng P
oint
Sect
ion
III -
Phy
sica
l/Che
mic
al C
hara
cter
istic
s
Unu
sual
Fir
e an
d Ex
plos
ion
Haz
ards
Spec
ial F
ire
Figh
ting
Proc
edur
es
Vapo
r Pr
essu
re (
mm
Hg.
)
Vapo
r D
ensi
ty (
AIR
= 1
)
Solu
bilit
y in
Wat
er
App
eara
nce
and
Odo
r
Sect
ion
IV -
Phy
sica
l/Che
mic
al C
hara
cter
istic
sFl
ash
Poin
t (M
etho
d U
sed)
Extin
guis
hing
Med
ia
Flam
mab
le L
imits
UEL
LEL
Mel
ting
Poin
t
Evap
orat
ion
Rat
e(B
utyl
Ace
tate
= 1
)
Spec
ific
Gra
vity
(H
0 =
1)
2
EDV
OT
EK®
Enzy
me
Rea
ctio
n Bu
ffer
10/1
0/06
Thi
s pr
oduc
t co
ntai
ns n
o ha
zard
ous
mat
eria
ls a
s de
fined
by
the
OSH
A H
azar
dC
omm
unic
atio
n St
anda
rd.
No
data
No
data
No
data
No
data
N/A
No
data
solu
ble
Cle
ar li
quid
, no
odor
, dry
che
mic
al, c
arbo
n di
oxid
e, w
ater
spr
ay o
r fo
am.
No
data
No
data
N
o da
ta
Use
ext
inqu
ishi
ng m
edia
app
ropr
iate
to
surr
ound
ing
fire
Rem
ove
cont
aine
r fr
om fi
re if
pos
sibl
e.
May
pro
duce
tox
ic g
ases
Stab
ility
Sect
ion
V -
Rea
ctiv
ity D
ata U
nsta
ble
Sect
ion
VI -
Hea
lth H
azar
d D
ata
Inco
mpa
tibili
ty
Con
ditio
ns t
o A
void
Rou
te(s
) of
Ent
ry:
Inha
latio
n?In
gest
ion?
Skin
?
Oth
er
Stab
le
Haz
ardo
us
Poly
mer
izat
ion
May
Occ
urC
ondi
tions
to
Avo
id
Will
Not
Occ
ur
Hea
lth H
azar
ds (
Acu
te a
nd C
hron
ic)
Car
cino
geni
city
:N
TP?
OSH
A R
egul
atio
n?IA
RC
Mon
ogra
phs?
Sign
s an
d Sy
mpt
oms
of E
xpos
ure
Med
ical
Con
ditio
ns G
ener
ally
Agg
rava
ted
by E
xpos
ure
Emer
genc
y Fi
rst A
id P
roce
dure
s
Sect
ion
VII
- Pr
ecau
tions
for
Safe
Han
dlin
g an
d U
seSt
eps
to b
e Ta
ken
in c
ase
Mat
eria
l is
Rel
ease
d fo
r Sp
illed
Was
te D
ispo
sal M
etho
d
Prec
autio
ns t
o be
Tak
en in
Han
dlin
g an
d St
orin
g
Oth
er P
reca
utio
ns
Sect
ion
VIII
- C
ontr
ol M
easu
res
Vent
ilatio
nLo
cal E
xhau
stSp
ecia
l
Mec
hani
cal (
Gen
eral
)
Res
pira
tory
Pro
tect
ion
(Spe
cify
Typ
e)
Prot
ectiv
e G
love
s
Oth
er P
rote
ctiv
e C
loth
ing
or E
quip
men
t
Wor
k/H
ygie
nic
Prac
tices
Eye
Prot
ectio
n
Haz
ardo
us D
ecom
posi
tion
or B
ypro
duct
s
Cop
per,
iron
, silv
er s
alts
, hyd
roge
n pe
roxi
de, p
heno
l, pi
cric
aci
dX X
Trea
t sy
mpt
omat
ical
ly a
nd s
uppo
rtiv
ely
Prot
ectio
n to
avo
id s
kin
cont
act
Non
e
Yes
Yes
Ye
sTo
xici
ty h
as n
ot b
een
quan
tifie
d. S
ensi
tivity
rea
ctio
ns (
alle
rgic
)
May
cau
se ir
rita
tion
to s
kin/
eye,
muc
ous
mem
bran
es a
nd u
pper
res
pira
tory
tra
ct.
Res
pira
tory
con
ditio
ns
Mop
up
with
abs
orba
nt m
ater
ial.
Dis
pose
of p
rope
rly.
Mix
with
ver
mic
ulite
and
dry
cau
stic
, wra
p in
pap
er a
nd b
urn
in a
che
mic
al in
cine
rato
r eq
uipp
ed w
ith
afte
rbur
ner
and
scru
bber
. Ig
nite
in p
rese
nce
of s
odiu
m c
arbo
nate
and
sla
ked
lime
(CaO
H)
Wea
r pr
otec
tive
gear
to
avoi
d sk
in/e
ye c
onta
ct
Non
e
Che
mic
al c
artr
idge
res
pira
tor
with
org
anic
vap
or c
artr
idge
.
Yes
N
one
No
Proc
ess
Encl
. Ven
t. Sy
s.
yes
Sp
lash
pro
of g
oggl
es
Do
not
inge
st. A
void
con
tact
with
ski
n, e
yes
and
clot
hing
. Was
h th
orou
ghly
af
ter
hand
ling.
Non
e
Form
alde
hyde
, eth
er, a
lcoh
ol, n
itrog
en o
xide
, str
ong
base
s, ox
idiz
ing
agen
ts.
may
occ
ur b
y sk
in p
enet
ratio
n in
clud
ing
anap
hyla
ctic
sho
ck.
Non
e
No
data
No
data
No
data
Toxi
c ga
ses:
Car
bon
mon
oxid
e, c
arbo
n di
oxid
e, n
itrog
en o
xide
s, ch
lori
ne, h
ydro
gen
chlo
ride
38
225EDVO-Kit # Material Safety Data Sheets
Full size (8.5 x 11”) pdf copy of MSDS available at www.edvotek.com or by request.
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
X X
Trea
t sy
mp
tom
atic
ally
an
d s
up
po
rtiv
ely
Rin
se c
on
tact
ed a
rea
wit
h c
op
iou
s am
ou
nts
of
wat
er.
No
ne
req
uir
ed
No
ne
Yes
Y
es
Y
es
May
cau
se s
kin
or
eye
irri
tati
on
No
ne
rep
ort
ed
Rin
se c
on
tact
ed a
rea
wit
h c
op
iou
s am
ou
nts
of
wat
er.
Ob
serv
e al
l fed
eral
, sta
te, a
nd
loca
l reg
ula
tio
ns.
Avo
id e
ye a
nd
ski
n c
on
tact
.
No
ne
Ch
emic
al c
artr
idg
e re
spir
ato
r w
ith
org
anic
vap
or
cart
rid
ge.
Yes
Yes
Yes
No
ne
yes
Sp
lash
pro
of
go
gg
les
Do
no
t in
ges
t. A
void
co
nta
ct w
ith
ski
n, e
yes
and
clo
thin
g.
Was
h t
ho
rou
gh
ly a
fter
han
dlin
g.
No
ne
No
ne
kno
wn
Sulf
ur
oxi
des
an
d b
rom
ides
Acu
te e
ye c
on
tact
: M
ay c
ause
irri
tati
on
N
o d
ata
avai
lab
le f
or
oth
er r
ou
tes
No
ne
No
dat
a
No
dat
a
N
o d
ata
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
Gel
load
ing
so
luti
on
co
nce
ntr
ate,
10x
10/1
0/06
This
pro
du
ct c
on
tain
s n
o h
azar
do
us
mat
eria
ls a
s d
efin
ed b
y th
e O
SHA
Haz
ard
Co
mm
un
icat
ion
Sta
nd
ard
.
No
dat
a
No
dat
a
No
dat
a
No
dat
a
N/A
No
dat
a
solu
ble
Blu
e liq
uid
, no
od
or
Un
kno
wn
No
dat
a
No
dat
a
No
dat
a
Dry
ch
emic
al, c
arb
on
dio
xid
e, w
ater
sp
ray
or
foam
Use
ag
ents
su
itab
le f
or
typ
e o
f su
rro
un
din
g f
ire.
Kee
p u
pw
ind
, avo
id
bre
ath
ing
haz
ard
ou
s su
lfu
r o
xid
es a
nd
bro
mid
es.
Wea
r SC
BA
.
ED
VO
TE
K®
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Iden
tify
Info
rmat
ion
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
ED
VO
TE
K®
Inst
aSta
in®
Met
hyl
ene
Blu
e, M
eth
ylen
e B
lue
Plu
s™
10/0
5/06
Met
hyl
ene
Blu
e
3.7
Bis
(D
imet
hyl
amin
o)
Phen
oth
iazi
n 5
IUM
C
hlo
rid
e
No
dat
a av
aila
ble
CA
S #
61-7
3-4
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
Solu
ble
- c
old
Ch
emic
al b
ou
nd
to
pap
er, n
o o
do
r
No
dat
a av
aila
ble
No
dat
a
N
o d
ata
Wat
er s
pra
y, c
arb
on
dio
xid
e, d
ry c
hem
ical
po
wd
er, a
lco
ho
l or
po
lym
er f
oam
Self
co
nta
ined
bre
ath
ing
ap
par
atu
s an
d p
rote
ctiv
e cl
oth
ing
to
pre
ven
t co
nta
ct
wit
h s
kin
an
d e
yes
Emit
s to
xid
fu
mes
un
der
fir
e co
nd
itio
ns
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
X
No
ne
Stro
ng
oxi
diz
ing
ag
ents
Toxi
c fu
mes
of
Car
bo
n m
on
oxi
de,
Car
bo
n d
ioxi
de,
n
itro
gen
oxi
des
, su
lfu
r o
xid
es, h
ydro
gen
, ch
lori
de
gas
X
N
on
e
Yes
Y
es
Yes
Skin
: M
ay c
ause
ski
n ir
rita
tio
n
Eyes
: M
ay c
ause
eye
irri
tati
on
In
hal
atio
n:
Cya
no
sis
Mee
ts c
rite
ria
for
pro
po
sed
OSH
A m
edic
al r
eco
rds
rule
PER
EAC
47.
3042
0.82
No
dat
a av
aila
ble
No
dat
a av
aila
ble
Trea
t sy
mp
tom
atic
ally
Ven
tila
te a
rea
and
was
h s
pill
sit
e
Mix
mat
eria
l wit
h a
co
mb
ust
ible
so
lven
t an
d b
urn
in c
hem
ical
inci
ner
ato
r eq
uip
ped
wit
h a
fter
bu
rner
an
d s
cru
bb
er.
Ch
eck
loca
l an
d s
tate
reg
ula
tio
ns.
Kee
p t
igh
tly
clo
sed
. St
ore
in c
oo
l, d
ry p
lace
No
ne
MIO
SH/O
SHA
ap
pro
ved
, SC
BA
Req
uir
ed
Ru
bb
erC
hem
. saf
ety
go
gg
les
Ru
bb
er b
oo
ts
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
(G
ener
al)
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
X
N
on
e
No
ne
Sulf
ur
oxi
des
, an
d b
rom
ides
X
N
on
e
Yes
Y
es
Yes
Acu
te e
ye c
on
tact
: M
ay c
ause
irri
tati
on
. N
o d
ata
avai
lab
le f
or
oth
er r
ou
tes.
No
dat
a av
aila
ble
May
cau
se s
kin
or
eye
irri
tati
on
No
ne
rep
ort
ed
Trea
t sy
mp
tom
atic
ally
an
d s
up
po
rtiv
ely.
Rin
se c
on
tact
ed a
rea
wit
h c
op
iou
s am
ou
nts
of
wat
er.
Wea
r ey
e an
d s
kin
pro
tect
ion
an
d m
op
sp
ill a
rea.
Rin
se w
ith
wat
er.
Ob
serv
e al
l fed
eral
, sta
te, a
nd
loca
l reg
ula
tio
ns.
Avo
id e
ye a
nd
ski
n c
on
tact
.
No
ne
Yes
No
ne
Yes
No
ne
Yes
Spla
sh p
roo
f g
og
gle
s
No
ne
req
uir
ed
Avo
id e
ye a
nd
ski
n c
on
tact
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
Prac
tice
Gel
Lo
adin
g S
olu
tio
n
10/0
5/06
This
pro
du
ct c
on
tain
s n
o h
azar
do
us
mat
eria
ls a
s d
efin
ed b
y th
e O
SHA
Haz
ard
Co
mm
un
icat
ion
Stan
dar
d.
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
No
dat
a
Solu
ble
B
lue
liqu
id, n
o o
do
r
No
dat
aN
o d
ata
No
dat
a
Dry
ch
emic
al, c
arb
on
dio
xid
e, w
ater
sp
ray
or
foam
Use
age
nts s
uita
ble
for t
ype
of su
rrou
ndin
g fir
e. K
eep
upw
ind,
avo
idb
reat
hin
g h
azar
do
us
sulf
ur
oxi
des
an
d b
rom
ides
. W
ear
SCB
A.
Un
kno
wn
ED
VO
TE
K®