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The Biotechnology Education Company ® • • www.edvotek.com
Sci-On Biology®
®
EVT 003195AM
All components are intended foreducational research only. Theyare not to be used for diagnosticor drug purposes, noradministered to or consumed byhumans or animals.
S-49EDVO-Kit #
In Search of My Father
Storage:Store this experiment at room temperature
EXPERIMENT OBJECTIVES:
Students will learn how agarose gel electro-phoresis separates different sizes of dyemolecules that represent DNA fragments.They will learn how these fragments formunique DNA patterns for each person,which is the basis for solving maternity andpaternity identity.
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EDVO-Kit # S-49 In Search of My Father2
EVT 003195AM
Page
Experiment Components 3
Experiment Requirements 3
Background Information 4
Experiment Procedures
Experiment Overview 6
Activity One - Agarose Gel Preparation
and Practice Gel Loading 7
Activity Two - Conducting Agarose Gel Electrophoresis 12
Critical Thinking and Hypothesis Development 14
Study Questions 14
Instructor's Guidelines
Notes to the Instructor 15
Suggestions for Lesson Plan Content 16
Connections to National Content and Skill Standards 17
Pre-Lab Preparations 18
Notes Regarding Electrophoresis 21
Experiment Results 22
Study Questions and Answers 23
Material Safety Data Sheets 24
Table of Contents
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EDVO-Kit # S-49 In Search of My Father3
EVT 003195AM
ELECTROPHORESIS SAMPLES
• Ready-to-Load™ Dye samplesA Standard dyes with assigned
base pair equivalentsB Mother 1 DNAC Mother 2 DNAD Boy 1 DNAE Boy 2 DNAF Father (surviving, married to Mother 1)
REAGENTS & SUPPLIES:
• Practice Gel Loading Solution• UltraSpec-Agarose™ powder• Concentrated electrophoresis buffer• 1 ml pipet• 100 ml graduated cylinder (packaging for samples)• Microtipped Transfer Pipets
Experiment Components
• 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
• DNA visualization system (white light)
• Distilled or deionized water
Requirements
All components areintended foreducational researchonly. They are not tobe used fordiagnostic or drugpurposes, noradministered to orconsumed byhumans or animals.
Storage:Store entire experimentat room temperature.
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
Using DNA Fingerprinting for Determining Familial Relationships
In a war torn country, two youngboys were separated from theirrespective parents who had beenimprisoned by the regime. Thechildren were cousins who wereborn two months apart and lookedstrikingly similar. Their mothers werehalf sisters who shared a commonmother. After several years, theregime was overthrown andreplaced by a new governmentthat released all political prisonersfrom prison. The prisoners werereinstated in their respectivecommunities and all chargesagainst them were dropped.
During this period, the two boys had not been separated from eachother, and had been adopted by a high ranking military officer whohad no children of his own. Both the officer and his wife died in aliberation uprising and for a short period of time the boys wereplaced in an orphanage. On their 18th birthday, the boys werereleased from the orphanage and they immediately began tosearch for information regarding their lost parents.
The brothers contacted a number of elders from their village. Theelders informed them that after their biological parents were re-leased from prison, one of the fathers was assassinated by individualswho were sympathetic to the fallen regime. The second biologicalfather was suffering from amnesia. Two women, who were thought tobe their biological mothers, were in a rehabilitation facility whichhoused approximately 200 women.
Upon arrival to the center it was very obvious that the patients hadnot received proper medical attention. Based on age and appear-ance, about 10 women fit the profile of the two mothers.
Since the boys were cousins and their mothers were sisters whoshared the same mother (who was the grandmother of the boys),the first identification screening test performed was a mitochondrialDNA fingerprinting. Mitochondrial information is inherited onlythrough the female (mother). It was expected that the mitochon-drial testing results would be identical for the two boys. Mitochon-
Grandma's First
Husband
Grandma
Mother 1
Child
Mito
chon
drial D
NA M
atch
Grandma's Second Husband
Child
Mitochondrial DNA M
atch
Mother 2
Father 1
Boy Cousins
Father 2
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Experiment P
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
Using DNA Fingerprinting for Determining Familial Relationships
drial DNA fingerprinting tests are also less expensive than chromo-somal DNA testing and results are available in a shorter period of time.Results showed that two women did have the same matching mito-chondrial DNA fingerprinting pattern which also matched that of thetwo boys.
In order to match the boys with the correct mothers and the survivingfather, chromosomal DNA fingerprinting tests were ordered for theboys, the mothers and the surviving father. The DNA fingerprint of oneof the boys matched with the surviving father and one of the mothers.The DNA fingerprint of a child is a composite of elements (matchingvisible DNA bands on the gel) from both parents. The pattern is easilyrecognizable and can be experimentally demonstrated in this simula-tion experiment.
Chromosomal deoxyribonucleic acid (DNA), present in the nucleus ofevery living cell, is the genetic material that acts as a blueprint for allof the proteins synthesized by that cell. In mammals, however, a largefraction of the total DNA does not encode protein and serves noobvious function. Polymorphic DNA refers to chromosomal regionsthat vary widely from individual to individual. By examining several ofthese regions within the genomic DNA obtained from an individual,one may determine a “DNA Fingerprint” for that individual. DNApolymorphisms are now widely used for determining paternity/maternity, kinship.
Mitochondrial DNA is inherited from the mother. A human eggcontains a large number of mitochondria. Unlike that, human spermcontains a small number of mitochondria. Upon fertilization of thehuman egg by a sperm, the zygote contains the majority of mito-chondria obtained by maternal genetic inheritance. Unlike mito-chondrial DNA, nuclear cell DNA is an equal composite of bothparents. In each chromosome pair, one is inherited from the fatherand the second from the mother.
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
BEFORE YOU START THE EXPERIMENT
1. Read all instructions before starting the experiment.
2. Write a hypothesis that reflects the experiment and predictexperimental outcomes.
Experiment Overview
Attach safety cover, connect leads to power source and
conduct electrophoresis
Remove end blocks, comb and submerge gel under buffer
in electrophoresis chamber
Prepare agarosegel in casting tray
Load each dye samplein consecutive wells.
WORKING HYPOTHESIS
If DNA samples collected fromdifferent mothers and fathers areexamined at variable polymorphicsites, then one should be able tomatch the children with their realmother and father by the DNAfingerprinting method.
EXPERIMENT CONTENT OBJECTIVE
Students will learn how agarose gel electro-phoresis separates different sizes of dyemolecules that represent DNA fragments.They will learn how these fragments formunique DNA patterns for each person, which isthe basis for solving maternity and paternityidentity.
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EDVO-Kit # S-49In Search of My Father
Experiment P
rocedures
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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
PREPARING THE GEL BED
1. Close off the open ends of a cleanand dry gel bed (casting tray) byusing rubber dams or tape.
A. Using Rubber dams:
• Place a rubber dam on eachend of the bed. Make surethe rubber dam fits firmly incontact 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 sidesand bottom edge of the bed.
• Fold the extended edges of the tape back onto the sidesand bottom. Press contact points firmly to form a goodseal.
2. Place a well-former template (comb)in the first set of notches at the end ofthe bed. Make sure the comb sitsfirmly and evenly across the bed.
Activity One - Agarose Gel Preparation and Practice Gel Loading
LABORATORY SAFETY
1. Gloves and goggles should be worn routinely as goodlaboratory practice.
2. Exercise extreme caution when working with equip-ment that is used in conjunction with the heatingand/or melting of reagents.
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4. Exercise caution when using any electrical equipment in thelaboratory.
5. Always wash hands thoroughly with soap and water after han-dling reagents or biological materials in the laboratory.
Wear glovesand safety
goggles
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
CASTING AGAROSE GELS
3. Use a 250 ml flask to prepare the gel solution. Add the followingcomponents to the flask as specified for your experiment (refer toTable A).
• Buffer concentrate• Distilled water• Agarose powder
Activity One - Agarose Gel Preparation and Practice Gel Loading
At high altitudes, it isrecommended to use amicrowave oven toreach boilingtemperatures.
4. Swirl the mixture to disperse clumps of agarose powder.
5. With a marking pen, indicate the level of the solution volume onthe outside of the flask.
6. Heat the mixture to dissolve the agarose powder. The finalsolution should appear clear (like water) without any undissolvedparticles.
A. Microwave method:
• Cover the flask with plastic wrap to minimize evapora-tion.
• 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 flask with aluminum foil to prevent excessevaporation.
• Heat the mixture to boiling over a burner with occasionalswirling. Boil until all the agarose is completely dissolved.
Check the solution carefully. If you see "crystal" particles, the agaroseis not completely dissolved.
Amt ofAgarose
(g)
ConcentratedBuffer (50x)
(ml)
Size of EDVOTEKCasting Tray
(cm)
DistilledWater(ml)
TotalVolume
(ml)
7 x 7
7 x 15
0.24
0.48
0.6
1.2
29.4
58.8
30
60
+ =+
Table AIndividual 0.8% UltraSpec-Agarose™ Gel
Electrophoresis of Dyes
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EDVO-Kit # S-49In Search of My Father
Experiment P
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
Activity One - Agarose Gel Preparation and Practice Gel Loading
7. Cool the agarose solution to 55°Cwith careful swirling to promoteeven dissipation of heat. Ifdetectable evaporation hasoccurred, add distilled water tobring the solution up to theoriginal volume as marked on theflask in step 5.
After the gel is cooled to 55°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 agar-ose solution from leaking.
• Use a transfer pipet to deposit a small amount of cooledagarose 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 thebed is on a level surface.
10. Allow the gel to completely solidify. It will become firm and coolto the touch after approximately 20 minutes.
Cool theagarose to
DO NOT POUR BOILINGHOT AGAROSE INTO THEGEL BED.
Hot agarose solution mayirreversibly warp the bed.
55˚C
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
PREPARING THE GEL FOR ELECTROPHORESIS
11. After the gel is completely solidified, carefully and slowly removethe rubber dams or tape from the gel bed.
Be especially careful not to damage or tear the gel wells when removingthe rubber dams. A thin plastic knife, spatula or pipet tip can be insertedbetween the gel and the dams to break possible surface tension.
12. Remove the comb by slowly pulling straight up. Do this carefullyand 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 requiredvolume of diluted buffer for the specific unit you are using (seeguidelines in Table B).
For DNA analysis, the same EDVOTEK 50x Electrophoresis Buffer is usedfor preparing both the agarose gel buffer and the chamber buffer. Theformula for diluting EDVOTEK (50x) concentrated buffer is 1 volume ofbuffer concentrate to every 49 volumes of distilled or deionized water.
Activity One - Agarose Gel Preparation and Practice Gel Loading
The electrophoresis(chamber) bufferrecommended is Tris-acetate-EDTA (20 mMtris, 6 mM sodiumacetate, 1 mM disodiumethylenediaminetetraacetic acid) pH 7.8.Prepare the buffer asrequired for yourelectrophoresisapparatus.
ConcentratedBuffer (50x)
(ml)
EDVOTEKModel #
DistilledWater(ml)
TotalVolume
(ml)
Table B Dilution of Electrophoresis(Chamber) Buffer
M6+
M12
M36 (blue)
M36 (clear)
6
8
10
20
294
392
490
980
300
400
500
1000
=+
15. Make sure the gel is completely covered with buffer.
16. Proceed to loading the samples and conducting electrophoresis.
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EDVO-Kit # S-49In Search of My Father
Experiment P
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
Activity One - Agarose Gel Preparation and Practice Gel Loading
If you are using transferpipets, gently squeeze thepipet stem, instead of thebulb to help control thedeliveryof smallsamplevolumes.
PRACTICE GEL LOADING
Accurate sample delivery technique ensures the best possible gelresults. Pipeting mistakes can cause the sample to become dilutedwith buffer, or cause damage to the wells with the pipet tip whileloading the gel.
If you are unfamiliar with loading samples in agarose gels, it is recom-mended that you practice sample delivery techniques beforeconducting the actual experiment. EDVOTEK® electrophoresisexperiments contain a tube of practice gel loading solution for thispurpose. Casting of a separate practice gel is highly recommended.One suggested activity is outlined below:
1. Cast a gel with the maximum number of wells possible.
2. After the gel solidifies, place it under buffer in an electrophoresisapparatus chamber.
Alternatively, your teacher may have cut the gel in sectionsbetween the rows of wells. Place a gel section with wells into asmall, shallow tray and submerge it under buffer or water.
Note: The agarose gel is sometimes called a "submarine gel" because it issubmerged under buffer for sample loading and electrophoretic separation.
3. Practice delivering the practice gel loading solution to thesample wells. Take care not to damage or puncture the wellswith the pipet tip.
• For electrophoresis of dyes, load the sample well with 35-38microliters of sample.
• If using transfer pipets for sample delivery, load each samplewell until it is full.
4. If you need more practice, remove the practice gel loadingsolution by squirting buffer into the wells with a transfer pipet.
5. Replace the practice gel with a fresh gel for the actual experi-ment.
Note: If practice gel loading is performed in the electrophoresis chamber,the practice gel loading solution will become diluted in the buffer in theapparatus. A small amount of practice gel loading solution (filling up to 12wells) will not interfere with the experiment, so it is not necessary to preparefresh buffer.
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
Activity Two - Conducting Agarose Gel Electrophoresis
ELECTROPHORESIS SAMPLES
Samples in this electrophoresis experimentare packaged in pre-aliquotedQuickstrip™ connected tubes.
To remove samples from theQuickstrip™ tubes, simply pierce thefoil top with the micropipet tip andwithdraw the sample.
EDVOTEKQuickstrips™
Quickstripspatent pending
LOADING THE SAMPLES
1. Check the Sample Volumes
Sometimes a small amount of sample will cling to the walls ofthe tubes. Make sure the entire volume of sample is at thebottom of the tubes before starting to load the gel.
• If your samples are in Quickstrip™ connectedtubes, tap the foil top of the strip so samplesfall to the bottom of the tubes.
• If your samples are in individual 1.5 ml or 0.5 mlmicrotest tubes, briefly centrifuge the sampletubes, or tap each tube on the tabletop toget all the sample to the bottom of the tube.
2. Load Samples
Load each of the dye samples in tubes A - F intothe wells in consecutive order. The amount ofsample that should be loaded is 35-38 µl.
Lane Label Sample
1 A Markers2 B Mother 13 C Mother 24 D Boy 15 E Boy 26 F Father
FEDCBA
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EDVO-Kit # S-49In Search of My Father
Experiment P
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
RUNNING THE GEL
3. After the samples are loaded, carefully snap the cover downonto the electrode terminals.
Make sure that the negative and positive color-coded indicators on thecover and apparatus chamber are properly oriented.
4. Insert the plug of the black wire into the black input of the powersource (negative input). Insertthe plug of the red wire into thered input of the power source(positive input).
5. Set the power source at therequired voltage and conductelectrophoresis for the length oftime determined by your instruc-tor. General guidelines arepresented in Table C.
6. Check to see that current isflowing properly - you should seebubbles forming on the twoplatinum electrodes.
Activity Two - Conducting Agarose Gel Electrophoresis
Table C Time andVoltage
RecommendedTime
Volts
125
70
50
20 min
40 min
60 min
Electrophoresis of Dyes
Staining is not required for Experiment # S-49, butresults must be analyzed upon completion of theelectrophoretic separation. Because dye moleculesare extremely small they will diffuse out of the gel.Thus, the gel cannot be saved.
7. After approximately 10 minutes,you will begin to see separationof the colored dyes.
8. After the electrophoresis iscompleted, turn off the power,unplug the power source,disconnect the leads andremove the cover.
9. Document the gel results.
A variety of documentationmethods can be used, includ-ing drawing a picture of the gel,taking a photograph, orscanning an image of the gelon a flatbed scanner.
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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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
Record the answers to the following Study Questions in your Labora-tory Notebook or as instructed by your teacher.
1. What do the different dye bands that were separated by electro-phoresis represent?
2. Why do different individuals, such as siblings, have differentfingerprints?
3. What is the basis of mitochondrial DNA fingerprint analysis?
4. What is the difference between mitochondrial and cell DNA-based fingerprinting analysis.
LABNOTEBOOK
Critical Thinking and Hypothesis Development
Record the following in your Laboratory Notebook or as instructed byyour teacher.
1. What is the variable in this experiment?
2. What is the control in this experiment?
3. What could one change in the experiment if this experiment wasrepeated?
4. Write a hypothesis that would reflect a change.
5. Based on the evidence obtained from the analysis of the gel,which child, mother and father are reunited? Explain.
LABNOTEBOOK
Study Questions
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Instructor’s Guide
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EVT 003195AM
EDVO-Kit # S-49In Search of My Father
Class size, length of laboratory sessions, and availability of equip-ment are factors which must be considered in the planning and theimplementation of this experiment with your students. These guide-lines include Suggestions for Lesson Plan Content which can beadapted to fit your specific set of circumstances.
APPROXIMATE TIME REQUIREMENTS
1. UltraSpec-Agarose™ gel preparation: Your schedule willdetermine when to prepare the gel(s) for an experiment.Whether you choose to prepare the gel(s) or have the studentsdo it, allow approximately 30-40 minutes for this procedure.Generally, 20 minutes of this time is required for gel solidifica-tion.
2. The approximate time for electrophoresis will vary from 20minutes to 60 minutes.
ELECTROPHORESIS HINTS AND HELP
EDVOTEK® Ready-to-Load Electrophoresis Experiments are easy toperform and are designed for maximum success in the classroomsetting. However, even the most experienced students and teach-
Notes to the Instructor
ers occasionally encounter experi-mental problems or difficulties.
The EDVOTEK® web site provides avariety of resources which are continu-ously being updated and added.Several suggestions and reminders forconducting electrophoresis areavailable, as well as answers tofrequently asked electrophoresisquestions.
www. edvotek.com
If you do not find the answers to yourquestions in this section or at theEDVOTEK® web site, Technical Serviceis available from 9:00 am to 6:00 pm,Eastern time zone. Call for help fromour knowledgeable technical staff at1-800-EDVOTEK (1-800-338-6835).
Technical ServiceDepartment
FAX: (301) 340-0582web: www.edvotek.comemail: [email protected]
Please have the following information:
• The experiment number and title• Kit Lot number on box or tube• The literature version number (in lower right corner)• Approximate purchase date
Mon - Fri9:00 am to 6:00 pm ET
Mon - Fr i 9 am
- 6pm
ET
1-800-EDVOTEK(1-800-338-6835)
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-TECH SERVICE
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EVT 003195AM
EDVO-Kit # S-49In Search of My Father
Suggestions for Lesson Plan Content
This teacher-generated lesson plan outline can be used as a guide-line for classroom discussion. Connections to the National Contentand Skills Standards follow the plan.
1. Have students write a creative scenario that is based on theanalysis of the gel, or divide students into small groups and haveeach group write a short play based on the evidence obtainedfrom the gel. Share scenarios with the class or give class time fora “short” play performance.
2. Using electrophoresis to separate DNA fragments as used in areasother than establishing biological relationships. Have studentsconduct an “On-line” search to see how this procedure is use inthe following cases:
• Identifying individuals that are carriers of genetic disease• Identifying men and women killed in service• Forensics
3. Discuss the importance of being meticulous in the collection andanalysis of DNA that will be used as evidence in court cases.
4. From the vocabulary list below, have students write applicationsentences about each:
DNA mitochondria nucleusElectrophoresis DNA Fingerprinting polymorphic DNA
Use pictures or drawings from textbooks and other resources tohelp explain the vocabulary words to the students.
DNA Fingerprinting is a method that allows for the matching andidentification of the source of unknown DNA samples. It canprovide positive identification with great accuracy. DNA finger-printing involves the electrophoretic analysis of DNA fragmentsizes obtained from a person’s DNA.
5. List and discuss with students the essential parts of an experiment.
• Writing a logical hypothesis• Making careful observations• Differentiating between an experiment and an control• Identifying variables• Predicting experimental outcomes• Recording results in a concise and accurate manner• Drawing valid interpretations of results• Formulating alternative explanations
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Instructor’s Guide
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EVT 003195AM
EDVO-Kit # S-49In Search of My Father
1. Students will develop abilities necessary to do scientific inquiry.• Student questions will be answered by conducting a scientific
investigation.
2. Students will develop an understanding through inquiry. They will:• Develop a logical hypothesis.• Make careful observations.• Interpret results correctly.• Understand the difference between the experiment and the
control• Identify and control the variable.• Predict experimental outcomes.• Formulate explanations from results.• Recognize and analyze alternative explanations.
3. Students will use equipment, materials, and techniques for experi-mentation and direct investigation of phenomena. They will:• Understand the principles of agarose gel electrophoresis.• Understand how different sizes of DNA fragments (as repre-
sented by dyes) are separated by agarose gel electrophoresis,resulting in unique DNA fingerprints for each individual.
4. Students will understand the principle behind DNA Fingerprinting• Students will understand that differences in DNA patterns
among individuals will result in unique DNA fingerprints foreach individual.
Connections to National Content Standards
Students will learn to load and run agarose gel electrophoresis. Analysisof the experiment will provide students the means to transform an ab-stract concept into a concrete explanation. Students will be able to:
1. Use scientific equipment such as calibrated pipets for metricmeasurements and run electrophoresis.
2. Accurately load and run an agarose gel.3. Make careful observations and record results.4. Accurately measure migration distances of known and unknown
fragments in a gel.5. Accurately plot data on semilog paper and determine fragment
sizes.
Connections to National Skill Standards
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’s G
uide
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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
EDVO-Kit # S-49In Search of My Father
BATCH AGAROSE GEL PREPARATION
To save time, the agarose gel solution can be prepared in a batch forsharing by the class. Any unused prepared agarose can be savedand remelted for gel casting at a later time. For a batch (375 ml)preparation of 0.8% agarose gel:
1. Use a 500 ml flask to prepare the diluted gel buffer.
• Add 7.5 ml of buffer concentrate
• Add 367.5 ml of distilled water.
2. Pour 3.0 grams of UltraSpec-Agarose™ into the prepared buffer.Swirl to disperse clumps.
3. With a marking pen, indicate the level of solution volume on theoutside of the flask.
4. Heat the agarose solution as previously described for individualgel preparation. The heating time will require adjustment due tothe larger total volume of gel buffer solution.
5. Cool the agarose solution to 55°C with swirling topromote even dissipation of heat. If evapora-tion has occurred, add distilled water to bringthe solution up to the original volume (375 ml) asmarked on the flask in step 3.
Pre-Lab Preparations
6. Dispense the required volume ofcooled agarose solution forcasting the gels. The volumerequired is dependent upon thesize of the gel bed (refer to TableA for individual gel castingguidelines).
7. Allow the gel to completelysolidify. It will become firm andcool to the touch after approxi-mately 20 minutes. Then proceedwith preparing the gel for electro-phoresis.
55˚C
Note: The UltraSpec-Agarose™ kit component isoften labeled with the amountit contains. In many cases, theentire contents of the bottle is3.0 grams. Please read the labelcarefully. If the amount ofagarose is not specified or ifthe bottle's plastic seal hasbeen broken, weigh the agaroseto ensure you are using thecorrect amount.
Amt ofAgarose
(g)
ConcentratedBuffer (50x)
(ml)
DistilledWater(ml)
TotalVolume
(ml)
3.0 7.5 367.5 375
+ =+
Table DBatch Preparation of
0.8% UltraSpec-Agarose™
®®
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Sci-On® Biology19
Instructor’s Guide
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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
EDVO-Kit # S-49In Search of My Father
Pre-Lab Preparations
ELECTROPHORESIS SAMPLES IN PRE-ALIQUOTEDQUICKSTRIP™ CONNECTED TUBES
The electrophoresis samples in this experiment are packaged in pre-aliquoted Quickstrip™ connected tubes.
1. Use sharp scissors to separate each set oftubes A-H in the block of samples.
Each row of samples (strip) constitutes acomplete set of samples for each gel. Thenumber of samples per set will varydepending on the experiment. Some tubesmay be empty.
2. Cut carefully through the foil between therows of samples. Do not cut or puncturethe foil covering the top of the sampletubes.
3. Each group will require one strip ofsamples.
4. Remind students to tap the foil or tubesbefore gel loading to ensure that all ofthe sample is at the bottom of the tube.
Carefully cut between
each set of tubes
ED
VO
TEK
®
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The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Sci-On® Biology
®®
20In
stru
ctor
’s G
uide
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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
EDVO-Kit # S-49In Search of My Father
MATERIALS FOR THE EXPERIMENT
Each Lab Group should have the following materials:
Activity One
• Agarose gel• Electrophoresis Buffer• Practice gel loading sample• Sample delivery instrument
Automatic micropipet and tips, orTransfer pipet and beaker of distilled water
Activity Two
• Agarose gel• Electrophoresis apparatus• DC power source• Dye Samples (A - F) representing DNA samples• Sample delivery instrument
Automatic micropipet and tips, orTransfer pipet and beaker of distilled water
Pre-Lab Preparations
®®
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Sci-On® Biology21
Instructor’s Guide
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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
EDVO-Kit # S-49In Search of My Father
Notes regarding electrophoresis
1. Do not move the apparatus immediately after the samples havebeen loaded.• Moving the apparatus will dislodge the samples from the wells
into the buffer and will compromise results.• If it is necessary to move the apparatus during electrophore-
sis, you may safely do so after the tracking dye has migratedat least 1 cm from the wells into the gel.
2. For optimal separation, do not use voltages higher than 125 voltsfor agarose gel electrophoresis. Higher voltages can overheatand melt the gel.
3. Electrophoresis should be terminated when the dyes have moved3 to 4 centimeters from the wells and before it moves off the gel.
AVOIDING COMMON PITFALLS
Potential pitfalls and/or problems can be avoided by following thesuggestions and reminders listed below.
• To ensure that dyes are well resolved, make sure the gel formula-tion is correct (see Table A) and that electrophoresis is conductedfor the optimal recommended amount of time.
• Correctly dilute the concentrated buffer for preparation of boththe gel and electrophoresis (chamber) buffer. Remember thatwithout buffer in the gel, there will be no sample mobility. Useonly distilled water to prepare buffers. Do not use tap water.
• For optimal results, use fresh electrophoresis buffer preparedaccording to instructions.
• Before performing the actual experiment, practice sampledelivery techniques to avoid diluting the sample with bufferduring gel loading.
• To avoid loss of samples into the buffer, make sure the gel isproperly oriented in the electrophoresis unit so the samples arenot electrophoresed in the wrong direction off the gel.
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Sci-On® Biology
®®
22In
stru
ctor
’s G
uide
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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
EDVO-Kit # S-49In Search of My Father
Experiment Results
Lane Tube
1 A Standard Dye Markers2 B Mother 13 C Mother 24 D Boy 15 E Boy 26 F Father
The purple band of Boy 2 matches the purpleband of Mother 1. The red band of Boy 2matches the red band of the father. There-fore, Boy 2 is the child of Mother 1 and thefather in this scenario.
Color legend
B1 Blue 1B2 Blue 2B3 Blue 3P Purple 1R RedY1 Yellow 1Y2 Yellow 2
In the two idealized schematics, the relative positions of dye molecules are shownbut are not depicted to scale.
Gel photo result
3,500
1,500
800
450
1 rehtoM
2 rehtoM
1 yoB2 yoB aF
rehtkra
M
sre
3,500
1,500
800
450
1 2 3 4 5 6
B1
Y1
B2
Y1
Y2R
PB3
Y2 R R
Y1P P
®®
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Sci-On® Biology23
Instructor’s Guide
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 purposewithout the written consent of EDVOTEK, Inc. Copyright © 2004 EDVOTEK, Inc., all rights reserved.
EVT 003195AM
EDVO-Kit # S-49In Search of My Father
Study Questions and Answers
1. What do the different dye bands that were separated by electro-phoresis represent?
The different dyes represent DNAs with variable lengths obtainedfrom the DNAs of the mother, child and fathers.
2. Why do different individuals, such as siblings, have individual(different) fingerprints?
Chromosomes which are matched pairs are derived one from thefather and the second from the mother. The two copies of agene at a specific focus of the chromosome represents theunique genotype for the particular offspring.
3. What is the basis of mitochondrial DNA fingerprint analysis?
Mitochondrial DNA is inherited from the mother. A human eggcontains a large number of mitochondria. Unlike that, humansperm contains a small number of mitochondria. Upon fertilizationof the human egg by a sperm, the zygote contains the majority ofmitochondria obtained by maternal genetic inheritance.
4. What is the difference between mitochondrial and cell DNA-based fingerprinting analysis.
Unlike mitochondrial DNA, nuclear cell DNA is an equal compos-ite of both parents. In each chromosome pair, one is inheritedfrom the father and the second from the mother.
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
®
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
Agarose
09-15-2002
This product contains no hazardous materials as defined by the OSHA Hazard CommunicationStandard.CAS #9012-36-6
For 1% solution 194° F
No data
No data
No data
No data
No data
Insoluble - cold
White powder, no odor
N.D. = No data
No data N.D. N.D.
Water spray, dry chemical, carbon dioxide, halon or standard foam
Possible fire hazard when exposed to heat or flame
None
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)Gen. dilution ventilation
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
Yes Splash proof goggles
Impervious clothing to prevent skin contact
None
X None
No data available
X None
Yes Yes Yes
Inhalation: No data available Ingestion: Large amounts may cause diarrhea
No data available
No data available
Treat symptomatically and supportively
Sweep up and place in suitable container for disposal
Normal solid waste disposal
None
None
Chemical cartridge respirator with full facepiece.
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
®
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
50x Electrophoresis Buffer
This product contains no hazardous materials as defined by the OSHA HazardCommunication Standard.
No data
No data
No data
No data
No data
No data
Appreciable, (greater than 10%)
Clear, liquid, slight vinegar odor
No data
N.D. = No data
N.D. N.D.
Use extinguishing media appropriate for surrounding fire.
Wear protective equipment and SCBA with full facepieceoperated in positive pressure mode.
None identified
09-17-2002
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
X None
Strong oxidizing agents
Carbon monoxide, Carbon dioxide
X None
Yes Yes Yes
None
None identified
Irritation to upper respiratory tract, skin, eyes
None
Ingestion: If conscious, give large amounts of water
Eyes: Flush with water Inhalation: Move to fresh air Skin: Wash with soap and water
Wear suitable protective clothing. Mop up spill
and rinse with water, or collect in absorptive material and dispose of the absorptive material.
Dispose in accordance with all applicable federal, state, and local enviromental regulations.
Avoid eye and skin contact.
None
Yes None
Yes None
Yes Safety goggles
None
None
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
®
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
Practice Gel Loading Solution
09-19-2002
This product contains no hazardous materials as defined by the OSHA Hazard CommunicationStandard.
No data
No data
No data
No data
No data
No data
Soluble
Blue liquid, no odor
No dataNo data No data
Dry chemical, carbon dioxide, water spray or foam
Use agents suitable for type of surrounding fire. Keep upwind, avoid
breathing hazardous sulfur oxides and bromides. Wear SCBA.
Unknown
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
X None
None
Sulfur oxides, and bromides
X None
Yes Yes Yes
Acute eye contact: May cause irritation. No data available for other routes.
No data available
May cause skin or eye irritation
None reported
Treat symptomatically and supportively. Rinse contacted area with copious amounts of water.
Wear eye and skin protection and mop spill area. Rinse with water.
Observe all federal, state, and local regulations.
Avoid eye and skin contact.
None
Yes None
Yes None
Yes Splash proof goggles
None required
Avoid eye and skin contact
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
NIOSH/MSHA - approved respirator
No None
No None
Yes Splash proof goggles
Wear eye and skin protection and mop/wipe spill area. Rinse with water.
Can be disposed in the trash or down the sink
Avoid eye and skin contact
None
Rinse contacted areas with copious amounts of water
X Unknown
None
X None
None required
Sulfur oxides and bromides
No Yes Yes
May cause skin or eye irritation
None reported
Avoid eye and skin contact
Acute eye contact: may cause irritation
None No data No data No
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
B-1: Food Dye
07/01/03
This product contains no hazardous materials as defined by the OSHA Hazard
Communication Standard.
No data
No data
No data
No data
N/A
No data
Soluble
Blue color, liquid, no odor
No data No data No data
N/A
N/A
None
EDVOTEK®
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
NIOSH/MSHA - approved respirator
No None
No None
Yes Splash proof goggles
Wear eye and skin protection and mop/wipe spill area. Rinse with water.
Can be disposed in the trash or down the sink
Avoid eye and skin contact
None
Rinse contacted areas with copious amounts of water
X Unknown
None
X None
None required
Sulfur oxides and bromides
No Yes Yes
May cause skin or eye irritation
None reported
Avoid eye and skin contact
Acute eye contact: may cause irritation
None No data No data No
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
B-2: Food Dye
07/01/03
This product contains no hazardous materials as defined by the OSHA Hazard
Communication Standard.
No data
No data
No data
No data
N/A
No data
Soluble
Blue color, liquid, no odor
No data No data No data
N/A
N/A
None
EDVOTEK®
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
®
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
B/101 Purple
09-16-2002
This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard
No data
No data
No data
No data
N/A
No data
Soluble
Blue liquid, no odor
No data No data No data
Dry chemical, carbon dioxide, water spray or foam.
Use agents suitable for type of surrounding fire. Keep upwind, avoid breathing hazardous sulfur oxides and bromides. Wear SCBA.
Unknown
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
X None
None
Sulfur oxides and bromides
X None
Acute eye contact: May cause irritation
------------------ No data ------------------
None required
Avoid eye and skin contact
Yes Yes Yes
May cause skin or eye irritation
None reported
Treat symptomatically and supportively. Rinse contacted areas with copious amounts
Wear eye and skin protection and mop/wipe spill area. Rinse with water.
Observe all federal, state, and local regulations.
Avoid eye and skin contact.
None
NIOSH/MSHA approved
Yes None
Yes None
Yes Splash proof goggles
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
®
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
Coomassie Blue
09-17-2002
CH3OHCAS # 67-56-1
65 C
96mmHg
1.11
.79
N/A
4.6
complete (100%)
Blue liquid/alcoholic, pungent odor
Use alcohol foam, dry chemical or carbon dioxide. (water may be ineffective)
Wear protective gear and SCBA w/ full facepiece operated in positive pressure mode.Move containers from fire area if possible.
Vapors may flow along surfaces to distant ignition sources and flash back.
(closed cup) 12C 6.0% 36.%
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
Chronic kidney damage, liver damage
Non-identified No No No
Skin/eye irritation, headache, nausea, vomiting, dizziness, narcosis, respirator, low blood pressure
Eye disorders, skin disorders, liver or kidney disorders
X
X
Faceshield, uniform, protective suit
Avoid contact
Yes Yes Yes
Inhalation: remove to fresh air Skin/eye contact: flush with copious amounts of waterIngestion: Call physician. If conscience give copious amts of water
Wear SCBA and full protective clothing. Shut off sources. Stop leak if possible withoutUse water spray to induce vapors. Use sand or non combustible absorbent material and sweep up
Dispose in accordance with all applicable federal, state, and local regulations
No flares, smoking, or flames in area. Keep container tightly closed.Store in a dry, well ventilated, flammable liquid storage area.
At concentrations above 200ppm, wear SCBA
Yes must meet TLV require None Yes None
Yes safety goggles
None
Heat, flames, other sources of ignition
Strong oxidizing agents, strong acids, zinc, aluminum, magnesium
Carbon monoxide, carbon dioxide, formaldehyde, nitrogen sulfur dioxide
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
NIOSH/MSHA - approved respirator
No NoneNo None
Yes Splash prof goggles
Wear eye and skin protection and mop/wipe spill area. Rinse with water.
Can be disposed in the trash or down the sink
Avoid eye and skin contact
None
Rinse contacted areas with copious amounts of water
X Unknown
None
X None
None required
Sulfur oxides and bromides
No Yes Yes
May cause skin or eye irritation
None reported
Avoid eye and skin contact
Acute eye contact: may cause irritation
None No data No data No
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
Methyl Orange
07/01/03
This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. CAS # 547-58-0
No data
No data
No data
No data
N/A
No data
Soluble
Yellow-orange color, liquid, no odor
No data No data No data
N/A
N/A
None
EDVOTEK®
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid ProceduresIngestion: Rinse mouth with water if person is conscious. Inhalation: Remove to fresh airSkin & eye contact: Rinse with copious amounts of water.
Wear protective clothing: Ventilate area and wipe with absorptive material. Dispose of absorptive material properly.
Mix with combustible solvent and burn in chemical incinerator equipped with afterburner and scrubber. Observe all federal, state, and local laws.
None
None
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
NIOSH/MSHA - approved respirator
No NoneYes None
Rubber Chem. safety goggles
X None
None
X None
Not thoroughly invvestigated
N/A
Rubber boots
Toxic fumes of carbon monoxide, carbon dioxide, nitrogen oxides and sulfur oxides.
Yes Yes Yes
No data
No data
Do not ingest. Avoid contact with skin, eyes and clothing.
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
®
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
A/101 Orange
09-18-2002
This product contains no hazardous materials as defined by the OSHA Hazard CommunicationStandard.
No data
No data
No data
No data
N/A
No data
Soluble
Orange liquid, no odor
No data No data No data
Water spray, carbon dioxide, dry chemical powder, alcohol or polymer foam.
Wear SCBA and protective clothing to prevent contact with skin and eyes.
Emits toxic fumes of carbon monoxide, carbon dioxide, nitrogen oxides and sulfur oxides under fire conditions.
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
®
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
C/101 Red
09-19-2002
Red CAS# 34487-61-1
No data
No data
No data
No data
285°C
No data
Water Soluble
Red powder
No data No data No data
Water spray, carbon dioxide, dry chemical powder, or appropriate foam.
Wear SCBA and protective clothing to prevent contact with skin and eyes.
Emits toxic fumes under fire conditions.
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid ProceduresFlush with copious amounts of H20 for at least 15 minutes . Remove to fresh air & remove contaminated clothing
Sweep up, place in a bag and hold for waste disposal. Avoid raising dust.Ventilate area and wash spill site after pickup.
See above
Store in a cool dry place
Avoid contact
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
NIOSH/MSHA - approved respirator
Yes NoneNo None
Chemical resistant Safety goggles
X Incompatibles
Strong oxidizing agents
X Unknown
Lab coat
CO, CO2, Sulfur Dioxide
Yes Yes Yes
Eye-skin irritation
No data
Do not ingest. Avoid contact with skin, eyes and clothing.
Stability
Section V - Reactivity DataUnstable
Section VI - Health Hazard Data
Incompatibility
Conditions to Avoid
Route(s) of Entry: Inhalation? Ingestion?Skin?
Other
Stable
Hazardous Polymerization
May Occur Conditions to Avoid
Will Not Occur
Health Hazards (Acute and Chronic)
Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?
Signs and Symptoms of Exposure
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled
Waste Disposal Method
Precautions to be Taken in Handling and Storing
Other Precautions
Section VIII - Control Measures
Ventilation Local Exhaust Special
Mechanical (General)
Respiratory Protection (Specify Type)
Protective Gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
Eye Protection
Hazardous Decomposition or Byproducts
NIOSH/MSHA - approved respirator
No NoneNo None
Yes Splash prof goggles
Wear eye and skin protection and mop/wipe spill area. Rinse with water.
Can be disposed in the trash or down the sink
Avoid eye and skin contact
None
Rinse contacted areas with copious amounts of water
X Unknown
None
X None
None required
Sulfur oxides and bromides
No Yes Yes
May cause skin or eye irritation
None reported
Avoid eye and skin contact
Acute eye contact: may cause irritation
None No data No data No
Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Section IManufacturer's Name
Section II - Hazardous Ingredients/Identify Information
Emergency Telephone Number
Telephone Number for information
Date Prepared
Signature of Preparer (optional)
Address (Number, Street, City, State, Zip Code)
EDVOTEK, Inc.
14676 Rothgeb DriveRockville, MD 20850
Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV
Other Limits Recommended % (Optional)
(301) 251-5990
(301) 251-5990
Boiling Point
Section III - Physical/Chemical Characteristics
Unusual Fire and Explosion Hazards
Special Fire Fighting Procedures
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Solubility in Water
Appearance and Odor
Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)
Extinguishing Media
Flammable Limits UELLEL
Melting Point
Evaporation Rate(Butyl Acetate = 1)
Specific Gravity (H 0 = 1) 2
Xylene Cyanol
07/01/03
This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. CAS # 2650-17-1
No data
No data
No data
No data
N/A
No data
Soluble
____________ color, liquid, no odor
No data No data No data
N/A
N/A
None
EDVOTEK®