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EVT 100202AM

EDVO-Kit

109DNA Fingerprinting:Identification ofDNA Restriction Fragmentation Patterns

See Page 3 for storage instructions.

ExPERImENT OBjECTIVE:

The objective of this experiment is to develop a basic understanding of DNA fingerprinting. Variations in restriction enzyme cleavage patterns

obtained from different DNA molecules will be analyzed and the possible perpetrator of a crime will be identified using

the logic of DNA fingerprinting.

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

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns

EVT 100202AM

All components are intended for educational research only. They are not to be used for diag-nostic or drug purposes, nor administered to or consumed by humans or animals.

THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experiment components are derived from human sources.

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

Page

Experiment Components 3

Experiment Requirements 3

Background Information 4

Experiment Procedures

Experiment Overview and General Instructions 10

Agarose Gel Electrophoresis 12

Study Questions 13

Instructor's Guidelines

Notes to the Instructor and Pre-Lab Preparations 15

Experiment Results and Analysis 21

Study Questions and Answers 22

Appendices 23

Material Safety Data Sheets 34

Table of Contents

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109Experiment


EVT 100202AM

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

FAx: (�01) �40-058� • email: [email protected]

READy-TO-LOAD™ DNA sAmPLEs FOR ELECTROPhOREsIs

A DNA from crime scene cut with Enzyme 1 B DNA from crime scene cut with Enzyme 2 C DNA from Suspect 1 cut with Enzyme 1 D DNA from Suspect 1 cut with Enzyme 2 E DNA from Suspect 2 cut with Enzyme 1 F DNA from Suspect 2 cut with Enzyme 2

REAgENTs & suPPLIEs

• UltraSpec-Agarose™ powder • Concentrated electrophoresis buffer • FlashBlue™ DNA Stain • InstaStain® Blue cards • Practice Gel Loading Solution • 1 ml pipet • Microtipped Transfer Pipets

Note: If you ordered Experiment #109-Q, the experiment components in-clude InstaStain® Ethidium bromide instead of FlashBlue™ and InstaStain® Blue DNA stains.

DNA samples are stable at room temperature. However, if the experiment will not be conducted within one month of receipt, it is recommended that the DNA samples be stored in the refrigerator.

DNA samples do not require heating prior to gel loading.

Experiment Components

Requirements

• Horizontal gel electrophoresis apparatus • D.C. power supply • Automatic micropipets with tips • Balance • Microwave, hot plate or burner • Pipet pump • 250 ml flasks or beakers • Hot gloves • Safety goggles and disposable laboratory gloves • Small plastic trays or large weigh boats (for gel destaining) • DNA visualization system (white light) • Distilled or deionized water

4

Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.

Copyright © 1989,1992,1994,1997,1998, 2000, 2004, 2007, 2009, EDVOTEK, Inc., all rights reserved. EVT 100202AM

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

DNA Fingerprinting - ID of DNA Restriction Fragmentation Patterns109 Experiment

Background Information

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

Human DNA fingerprinting was pioneered by Dr. Alex Jeffreys at the University of Leices-ter in 1984 which led to the apprehension of a murderer in the first DNA fingerprinting conviction in September 1987 in the UK. Two months later, the first U.S. conviction based on DNA fingerprinting occurred in Orlando, Florida. Since then, the use of DNA finger-printing has led to thousands of criminal convictions, as well as dozens of exonerations.

In contrast to earlier methodologies, such as blood typing which can only exclude a sus-pect, DNA fingerprinting can provide positive identification with great accuracy. In addi-tion to criminal identification cases, DNA fingerprinting is now used routinely in paternity determinations and for the identification of genetic disease "markers". It is also used for the identification of human remains, such as in war casualties, and was used extensively to identify victims of the September 11, 2001 terrorist attacks on the World Trade Cen-ter, the Pentagon, and passengers in the plane which crashed in a field near Shanksville, Pennsylvania.

Human cells contain two types of DNA. The first type is cellular chromosomal DNA, which is packaged in 23 sets of chromosomes in the nucleus of the cell. This DNA, obtained from both parents, reflects the combined parental genetic inheritance of an individual. DNA fingerprinting utilizing cellular DNA involves analysis of the sequence of two alleles for a particular gene.

The second type of DNA is different from cellular DNA and is present only in the mito-chondria, which are the energy-producing organelles of the cell. Mitochondrial DNA is inherited maternally by both males and females and is extremely useful in the analysis of specific cases where fraternal linkages are important to determine. For example, a broth-er, sister, half brother or half sister who share the same mother would inherit the same mitochondrial DNA. Identification is determined by sequencing certain regions within mitochondrial DNA, which is a single circular chromosome composed of 16,569 base pairs.

DNA fingerprinting developed by Dr. Jeffreys utilizes cellular chromosomal DNA submit-ted to restriction enzyme digestion and Southern blot analysis. When human DNA is digested by a restriction enzyme, a very large number of DNA fragments are generated. When separated by agarose gel electrophoresis, the numerous DNA fragments appear as a "smear" on the gel. Labeled probes are used to detect Restriction Fragment Length Polymorphic (RFLP) regions within DNA, which will be described in greater detail. DNA RFLP analysis is statistically very accurate but requires relatively large amounts of DNA and takes several days to perform.

In recent years, the use of the RFLP method has been overtaken by the Polymerase Chain Reaction (PCR) method because of two important advantages. The first is the sensitivity of PCR, which allows for DNA fingerprinting identification using much smaller amounts of DNA. This is because PCR is able to amplify DNA to facilitate analysis. The second ad-vantage is the speed of PCR analysis, which allows critical questions to be answered more quickly compared to Southern Blot analysis. One PCR cycle has three steps, resulting in a doubling of the amount of DNA (see Figure 1).

5

Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1989,1992,1994,1997,1998, 2000, 2004, 2007, 2009, EDVOTEK, Inc., all rights reserved. EVT 100202AM


109Experiment



The Polymerase Chain Reaction (PCR) method amplifies target sequences of DNA, which are referred to as AMRFLPs. PCR made it possible for very small amounts of DNA found at crime scenes to be amplified for DNA fingerprinting analysis. A specific set of two primers is used to prime DNA polymerase to synthesize many copies of the targeted areas of DNA.

Many important concepts of molecular biology can be conveyed in the context of DNA Fingerprinting methods. In this experiment, emphasis is placed on concepts related to RFLP analysis. The experiment activities will focus on the identification of DNA by ana-lyzing restriction fragmentation patterns separated by agarose gel electrophoresis.

3' 5' 5' 3'

5' 3' 3' 5'

5' 3'

5' 5'

Denature 94°C

Extension 72°C

3' 5'

Separation of 2 DNA strands

=

Primer 1 =

Primer 2 =

5' 3' 5'

Anneal 2 primers

45°C

3' 5' 5'

Cyc

le 1

Target Sequence

Figure 1: The Polymerase Chain Reaction

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usE OF REsTRICTION ENzymEs IN DNA FINgERPRINTINg

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

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

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

DNA is highly polymorphic - that is, no two individuals have exactly the same pattern of restriction enzyme recognition sites in their DNAs. A large number of al-leles exist in the population. Alleles, which are alter-nate forms of a gene, result in alternative expressions of genetic traits which can be dominant or recessive.

Chromosomes occur in matching pairs, one of maternal and the other of paternal origin. The two copies of a gene (alleles) at a given chromo-somal locus represent a composite of the parental genes constituting an individual’s unique genotype. It follows that alleles have differences in their base sequences which consequently creates differences in the distribution and frequencies of restriction en-zyme recognition sites. Other differences in base sequences between individuals can occur because of mutations and deletions. Such changes can also create or eliminate a recognition site.

Polymorphic DNA refers to chromosomal regions that vary widely from individual to in-dividual. By examining several of these regions within the genomic DNA obtained from an individual, one may obtain a "DNA fingerprint" for that individual. The most com-monly used polymorphisms are those that vary in length; these are known as Fragment Length Polymorphisms (FLPs). The main reason for the occurrence of RFLPs is because of variations in length of a given segment of genomic DNA between two restriction enzyme recognition sites among individuals of the same species.

5'....GGCC....3'3'....CCGG....5'

↑

↓Hae III

5'....GGATCC....3'3'....CCTAGG....5'

↑

↓Bam HI

5'....CTGCAG....3'3'....GACGTC....5'

↑

↓Pst I

5'....GANTC....3'3'....CTNAG....5'

↑

↓Hinf I

Figure 2: Restriction enzyme recognition sites

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109Experiment


Likewise, RFLP can occur in "intergenic" or noncoding regions of DNA and is known as Variable Number of Tandem Repeats (VNTRs). In this case, segments of DNA that contain sequences from 2 to 40 bases in length repeat in tandem manner many times. The num-ber of segments or "core unit" repeats varies among individuals of the same species while the restriction enzyme cut sites are not altered. VNTR loci are very polymorphic. There are potentially hundreds of alleles at a single locus and therefore they are very useful in DNA fingerprinting. Ten to fifteen percent of mammalian DNA consists of sets of repeat-ed, 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 sequences in DNA are flanked by recognition sites, the length of the repeat will determine the size of the restriction enzyme fragment generated. There are several types of these short, repetitive sequences and they have been characterized.

DNA FINgERPRINTINg usINg sOuThERN BLOTs

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

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


8





Analysis of the blotted DNA is done by hybrid-ization with a labeled DNA probe. In forensic RFLP analysis, the probe is a DNA fragment that contains base sequences which are complemen-tary to the variable arrays of tandemly repeated sequences found in the human chromosomes. Probes can be labeled with isotopic or non-iso-topic reporter molecules, such as fluorescent dyes 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 repeated sequences. The membrane is then washed to remove excess probe. If the probe is isotopically labeled to the membrane, it is then placed on an x-ray film for several hours. This process is known as autoradi-ography. Only DNA fragments that have hybrid-ized to the probe will reveal their positions on the film because the localized areas of radioac-tivity cause exposure. The hybridized fragments appear as discrete bands (fingerprint) on the film and are in the same relative positions as they were in the agarose gel after electrophoresis. Only specific DNA fragments, of the hundreds of thousands of fragments present, will hybridize with the probe because of the selective nature of the hybridization (base pairing) process.

In forensic cases, DNA samples can be extracted and purified from small specimens of skin, blood, semen, or hair roots collected at the crime scene. DNA that is suitable for analysis can also be obtained from dried stains of semen and blood. The RFLP analyses performed on these samples is then compared to samples obtained from the suspect. If the RFLP patterns match, it is then beyond reasonable doubt that the suspect was at the crime scene. In practice, several different probes containing different types of repetitious sequences are used in the hybridizations in order to satisfy certain statistical criteria for absolute, positive identification. To assure positive iden-tification in criminal cases, 13 different loci are compared between a suspect and evidence DNA obtained from the crime scene.

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

Lane 1 DNA Marker Lane 2 Homozygous Copies Lane 3 Heterozygous VNTR Lane 4 Heterozygous VNTR Lane 5 Homozygous Copies Lane 6 Heterozygous VNTR Lane 7 Homozygous Copies

(Lanes 3, 4, and 6 represent different combina-tions of the three VNTRs.)

Probe

Probe

Probe

A

B

C

1 2 3 4 5 6 7

30 Kb

40 Kb

50 Kb

50 Kb

40 Kb30 Kb A

B

C

Genotypes

Figure 3: RFLP analysis demonstrating Variable Numbers of Nucleotide Tandem Repeats (VNTR).


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109Experiment


In this experiment, DNAs are pre-digested by restriction enzymes and the fragmentation patterns serve as the individual fingerprint. The DNA fragmentation patterns can be analyzed directly in the stained agarose gel, which eliminates the need for a Southern blot. In this hypothetical case, DNA obtained from two suspects are cleaved with two restriction enzymes in separate reactions. The objective is to analyze and match the DNA fragmentation patterns after agarose gel electrophoresis and determine if Suspect 1 or Suspect 2 was at the crime scene.

THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA.

4

5

8

7 Autoradiograph

2

3

1

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. Blot DNA onto 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

Figure 4 DNA Fingerprinting by RFLP Analysis


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Exp

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Pro

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Experiment Overview and general Instructions

ExPERImENT OBjECTIVE:

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

LABORATORy sAFETy

1. Gloves and goggles should be worn routinely as good laboratory practice.

2. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents.

3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.

4. Exercise caution when using any electrical equipment in the laboratory.

5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory.

LABORATORy NOTEBOOK RECORDINgs:

Address and record the following in your laboratory notebook or on a separate worksheet.

Before starting the Experiment:

• Write a hypothesis that reflects the experiment. • Predict experimental outcomes.

During the Experiment: • Record (draw) your observations, or photograph the results.

Following the Experiment: • Formulate an explanation from the results. • Determine what could be changed in the experiment if the experiment were repeated. • Write a hypothesis that would reflect this change.

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109Experiment

DNA Fingerprinting - ID of DNA Restriction Fragmentation PatternsExp

erimen

t Proced

ure

After electrophoresis, transfer gel for staining

Analysis on white

light source

FlashBlue™DNA stain

Attach safety cover,connect leads to power

source and conduct electrophoresis

Load eachsample in

consecutive wells

Remove end blocks & comb, then submerge

gel under buffer in electrophoresis

chamber

Prepare agarose gel in

casting tray

6

5

4

3

2

1

Gel pattern will vary depending upon experiment.

Experiment Overview: Flow Chart

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Exp

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Pro

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Prepare the gel

1. Prepare an agarose gel with specifications summarized below. Your instructor will specify which DNA stain you will be using.

• Agarose gel concentration required: 0.8%

• Recommended gel size: 7 x 7 cm or 7 x 14 cm (two gels)

• Number of sample wells required: 6

• Placement of well-former template: first set of notches ( 7 x 7 cm) first & third set of notches (7 x 14 cm)

Reminders:

During electrophoresis, the DNA samples migrate through the agarose gel to-wards the positive electrode. Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.

+Black Red

Sample wells

–

Agarose gel Electrophoresis

Lane Tube A DNA from crime scene cut with Enzyme 1B DNA from crime scene cut with Enzyme 2C DNA from Suspect 1 cut with Enzyme 1D DNA from Suspect 1 cut with Enzyme 2E DNA from Suspect 2 cut with Enzyme 1F DNA from Suspect 2 cut with Enzyme 2

Run the gel

3. After DNA samples are loaded, connect the apparatus to the D.C. power source and set the power source at the required voltage.

4. Check that current is flowing properly - you should see bubbles forming on the two platinum electrodes. Conduct electrophoresis for the length of time specified by your instructor.

5. After electrophoresis is completed, proceed to DNA staining and visualiza-tion. Refer to Appendix E, F, G, or H for the appropriate staining instruc-tions.

6. Document the results of the gel by photodocumentation.

Alternatively, place transparency film on the gel and trace it with a permanent marking pen. Remember to include the outline of the gel and the sample wells in addition to the migration pattern of the DNA bands.

For gels to be stained with FlashBlue™ or InstaStain® Blue, prepare gels accord-ing to Appendix A.

For gels to be stained with InstaStain® Ethidium bromide, prepare gels ac-cording to Appendix B.

Step-by-step guidelines for agarose gel prepara-tion are summarized in Appendix D.

Wear Gloves & goggles

Load the samples

2. Load the DNA samples in tubes A - F into the wells in consecutive order.

• For gels to be stained with FlashBlue™ or InstaStain® Blue, fill wells with 35 - 38 µl.

• For gels to be stained with InstaStain® Ethidium Bromide, fill wells with 18 - 20 µl.

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109Experiment


1. Define FLP’s and give their significance.

�. What is the most likely cause of Restriction Fragment Length Polymorphisms?

�. What are Variable Number of Tandem Repeats (VNTRs)?

4. Who are the only individuals possessing the same DNA fingerprints?

5. List the steps involved in DNA fingerprinting from extraction of DNA through the matching of a suspect to a crime scene sample.

�. What type of human cells can be utilized for this technique?

study Questions

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EVT 100202AM

15

109Experiment


EVT 100202AM



Instructor’s guide

Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in planning and implementing this experi-ment with your students. These guidelines can be adapted to fit your spe-cific set of circumstances. If you do not find the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at 1-800-EDVOTEK (1-800-338-6835).

EDuCATIONAL REsOuRCEs, NATIONAL CONTENT AND sKILL sTANDARDs

By performing this experiment, students will learn to load samples and run agarose gel electrophoresis. Experiment analysis will provide students the means to transform an abstract concept into a concrete explanation.

Notes to the Instructor & Pre-Lab Preparations

Visit our web site for information about EDVOTEK's complete line of experiments for biotechnology

and biology education.

EDVOTEK Ready-to-Load Electrophoresis Ex-periments are easy to perform and are designed for maximum success in the classroom setting. However, even the most experienced students and teachers occasionally encounter experimen-tal problems or difficulties. EDVOTEK web site resources provide suggestions and valuable hints for conducting electrophoresis, as well as answers to frequently asked electrophoresis questions.

Laboratory Extensions and supplemental Activities

Laboratory extensions are easy to perform using EDVOTEK experiment kits. For example, a DNA sizing determination activity can be performed on any electrophoresis gel result if DNA markers are run in parallel with other DNA samples. For DNA Sizing instructions, and other laboratory ex-tension suggestions, please refer to the EDVOTEK website.

Visit the EDVOTEK web site often for continuously updated information.

Mon - Fri 9 am - 6 pm ET

(1-800-338-6835)

EDVO-TECH SERVICE

1-800-EDVOTEK

Mon - Fri9:00 am to 6:00 pm ET

FAX: (301) 340-0582Web: www.edvotek.comemail: [email protected]

Please have the following information ready:

• Experiment number and title• Kit lot number on box or tube• Literature version number (in lower right corner)• Approximate purchase date

Technical ServiceDepartment

OrderOnline

1�




109Experiment

Inst

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eInstructor’s Guide DNA Fingerprinting - ID of DNA

Restriction Fragmentation Patterns

APPROxImATE TImE REQuIREmENTs

1. Gel preparation: Whether you choose to prepare the gel(s) in advance or have the students prepare

their own, allow approximately 30 minutes for this procedure. Generally, 20 minutes of this time is required for gel solidification.

2. Micropipeting and Gel Loading: If your students are unfamiliar with using micropipets and sample loading tech-

niques, a micropipeting or practice gel loading activity is suggested prior to conduct-ing the experiment. Two suggested activities are:

• EDVOTEK Expt. # S-44, Micropipetting Basics, focuses exclusively on using micro-pipets. Students learn pipeting techniques by preparing and delivering various dye mixtures to a special Pipet Card™.

• Practice Gel Loading: EDVOTEK Series 100 electrophoresis experiments contain a tube of practice gel loading solution for this purpose. It is highly recommended that a separate agarose gel be cast for practice sample delivery. This activity can require anywhere from 10 minutes to an entire laboratory session, depending upon the skill level of your students.


3. Conducting Electrophoresis: The approximate time for electrophoresis will vary from

approximately 15 minutes to 2 hours. Different models of electrophoresis units will separate DNA at different rates depending upon its design configuration. Generally, the higher the voltage applied the faster the samples migrate. However, maximum voltage should not exceed the indicated recommendations. The Table C example at left shows Time and Voltage recommendations. Refer to Table C in Appendi-ces A or B for specific experiment guidelines.

PREPARINg AgAROsE gELs FOR ELECTROPhOREsIs

There are several options for preparing agarose gels for the electrophoresis experiments:

1. Individual Gel Casting: Each student lab group can be responsible for casting their own individual gel prior to conducting the experiment.

2. Batch Gel Preparation: A batch of agarose gel can be prepared for sharing by the class. To save time, a larger quantity of UltraSpec-Agarose can be prepared for shar-ing by the class. See instructions for "Batch Gel Preparation".

3. Preparing Gels in Advance: Gels may be prepared ahead and stored for later use. Solidified gels can be stored under buffer in the refrigerator for up to 2 weeks.

Do not store gels at -20°C. Freezing will destroy the gels.

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 Model

M6+ M12 & M36

Minimum / Maximum

25 / 35 min

35 / 45 min

60 / 90 min

95 / 130 min

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109Experiment

DNA Fingerprinting - ID of DNARestriction Fragmentation Patterns

Instru

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eInstructor’s Guide

* 0.77 UltraSpec-Agarose™ gel percentage rounded up to 0.8%

Amt ofAgarose

(g)

ConcentratedBuffer (50x)

(ml)

Size of Gel(cm)

DistilledWater(ml)

TotalVolume

(ml)

7 x 7

7 x 10

7 x 14

0.23

0.39

0.46

0.6

1.0

1.2

29.4

49.0

58.8

+ =+

Individual 0.8%* UltraSpec-Agarose™ Gel

DNA Staining with FlashBlue™ or InstaStain® Blue

Table

A.1

30

50

60

Table

A.2

Amt ofAgarose

(g)

DilutedBuffer (1x)

(ml)

Size of Gel(cm)

7 x 7

7 x 10

7 x 14

0.23

0.39

0.46

30

50

60

+



If preparing a 0.8% gel with concentrated (50x) buffer, use Table A.1

If preparing a 0.8% gel with diluted (1x) buffer, use Table A.2

usINg AgAROsE gELs ThAT hAVE BEEN PREPARED IN ADVANCE

If gels have been removed from their trays for storage, they should be "anchored" back to the tray with a few drops of hot, molten agarose before placing the gels into the apparatus for electrophoresis. This will prevent the gel from sliding around in the tray and/or floating around in the electrophoresis chamber.

AgAROsE gEL CONCENTRATION AND VOLumE

Gel concentration is one of many factors which affect the mobility of molecules during electrophoresis. Higher percentage gels are sturdier and easier to handle. However, the mobility of molecules and staining will take longer because of the tighter matrix of the gel. Gel volume varies depending on the size of the casting tray, as well as the type of stain to be used for DNA staining after electrophoresis. Gels which will be stained with InstaStain® Ethidium Bromide require less sample amount (volume) than gels that will be stained with FlashBlue™ or InstaStain® Blue.

This experiment requires a 0.8% gel. It is a common agarose gel concentration for sepa-rating dyes or DNA fragments in EDVOTEK experiments.

• Specifications for preparing a 0.8% gel to be stained with FlashBlue™ or In-staStain® Blue can be found in Appendix A.

• Specifications for preparing a 0.8% gel to be stained with InstaStain® Ethidium bromide can be found in Appendix B.

Tables A-1 and A-2 below are examples of tables from Appendix A. The first (left) table shows reagent volumes using concentrated (50x) buffer. The second (right) table shows reagent volumes using diluted (1x) buffer.


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109Experiment

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gEL sTAININg AND DEsTAININg AFTER ELECTROPhOREsIs DNA stains FlashBlue™ and InstaStain® Blue are included in EDVOTEK standard Series 100 experiments. For Series 100-Q experiments, InstaStain® Ethidium Bromide (InstaStain® EtBr) is included. InstaStain® is a proprietary staining method which saves time and reduces liquid waste. EDVOTEK also offers Protein InstaStain® for staining Protein polyacrylamide gels, which can be purchased separately.

Instructions for DNA staining options are provided in the Appendices section.

Option 1: FlashBlue™ liquid - Appendix E.

This simple and rapid liquid staining and destaining procedure yields excellent visibil-ity of DNA bands in less than 25 minutes (5 minutes staining, 20 minutes destaining).

Option �: Instastain® Blue cards, One-step staining and Destaining- Appendix F.

Agarose gels can be stained and destained in one easy step.

Option �: Instastain® Blue cards - Appendix g.

Using InstaStain® Blue cards, staining is completed in approximately 5-10 minutes. DNA bands will become visible after destaining for approximately 20 minutes. Results will become sharper with additional destaining. For the best photographic results, allow the gel to destain for several hours to overnight. This will allow the stained gel to "equilibrate" in the destaining solution, resulting in dark blue DNA bands contrasting against a uniformly light blue background.

Option 4: Instastain® Ethidium Bromide - Appendix h

Staining with ethidium bromide is very sensitive and can detect as little as 5 to 10 nanograms of DNA with the use of a U.V. transilluminator. Ethidium Bromide is a dye that is commonly used by scientific researchers. It is a listed mutagen and forms a tight complex with DNA by intercalating between the bases within the double helix. The complex strongly fluoresces when exposed to ultraviolet light.

CAUTION: Ethidium Bromide is a listed mutagen. Disposal of the InstaStain® EtBr cards, which contain microgram amounts of ethidium bromide, is minimal compared to the large volume of liquid waste generated by traditional ethidium bromide stain-ing procedures. Disposal of InstaStain® cards and gels should follow institutional guidelines for chemical waste.


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109Experiment


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READy-TO-LOAD DNA sAmPLEs FOR ELECTROPhOREsIs

No heating required before gel loading. EDVOTEK offers the widest selection of electrophoresis experiments which minimize expensive equipment requirements and save valuable time for integrating important biotechnology concepts in the teaching laboratory. Series 100 experiments feature DNA samples which are predigested with restriction enzymes and are stable at room temperature. DNA samples are ready for immediate delivery onto agarose gels for electrophoretic separation and do not require pre-heating in a waterbath.

Electrophoresis samples and reagents in EDVOTEK experiments are packaged in various formats. The samples in Series 100 and S-series electrophoresis experiments will be packaged in one of the following ways:

1) Pre-aliquoted Quickstrip™ connected tubes OR 2) Individual 1.5 ml (or 0.5 ml) microtest tubes

sAmPLEs FORmAT: PRE-ALIQuOTED QuICKsTRIP™ CONNECTED TuBEs

Convenient QuickStrip™ connected tubes contain pre-aliquoted ready-to-load samples. The samples are packaged in a microtiter block of tubes covered with a protective overlay. Separate the microtiter block of tubes into strips for a complete set of samples for one gel.

1. Use sharp scissors to separate the block of samples into individual strips as shown in the diagram at right.

Each row of samples (strip) constitutes a complete set of samples for each gel. The number of samples per set will vary depending on the experiment. Some tubes may be empty.

2. Cut carefully between the rows of samples. Do not cut or puncture the protective overlay directly covering the sample tubes.

FEDCBA

Carefully cut betweeneach set of tubes

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OTE

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3. Each gel will require one strip of samples.

4. Remind students to tap the tubes before gel loading to ensure that all of the sample is at the bottom of the tube.


�0




109Experiment

Inst

ruct

or’

s g

uid




sAmPLEs FORmAT: INDIVIDuAL 1.5 mL mICROTEsT TuBEs

It is recommended that samples packaged in 1.5 ml individual microtest tubes be aliquot-ed for each gel. DNA Samples packaged in this format are available in three standard quantities:

Standard experiment kit 240 µl Bulk B-Series 480 µl Bulk C Series 960 µl

1. Check all sample volumes for possible evaporation. Samples will become more con-centrated if evaporation has occurred.

2. If needed, tap or centrifuge the sample tubes. Then add distilled water to slightly above the following level:

1.3 cm level for Standard experiment kit 1.9 cm level for the B-Series 2.8 cm level for the C-Series

3. Mix well by inverting and tapping the tubes several times.

4. After determining that the samples are at their proper total volumes, aliquot each sample into appropriately labeled 0.5 ml or 1.5 ml microtest tubes.

• For gels to be stained with Flash-Blue™ or InstaStain® Blue:

35-38 µl of each sample

• For gels to be stained with In-staStain® Ethidium bromide:

18-20 µl of each sample

4.5

cm

B Ser

ies

4

80 µ

lC S

erie

s

9

60 µ

l

Expt.

Kit

2

40 µ

l

1.5 cm tubeApproximate

VolumeMeasurements

1.3

cm

1.9

cm 2.8

cm

Custom bulk quantities are also available by request.

5. If students have difficulty retrieving the entire aliquoted volume of sample because some of it clings to the side walls of the tubes, remind students to make sure all of the sample is at the bottom of the tube before gel loading. They should centrifuge the samples tubes, or tap the tubes on the tabletop.

�1



109Experiment


Instru

ctor’s g

uid


Experiment Results and Analysis

In the idealized schematic, the rela-tive positions of DNA fragments are shown but are not depicted to scale.

ANALysIs

• Lanes 1 and 2 (set one) represent the crime scene DNA digested by two different restriction enzymes, which yield distinctly different DNA band-ing patterns.

• Lanes 3 and 4 (set two) represent DNA from Suspect 1. The suspect's DNA has been digested with the same two restriction enzymes as in Lanes 1 and 2.

• Lanes 5 and 6 (set three) represent DNA from Suspect 2. It also has been digested with the same two enzymes as the crime scene DNA (Lanes 1 and 2) and DNA from Suspect 1 (Lanes 3 and 4).

Discussion Question:

Could these DNA samples have been distinguished from one another if only Enzyme 1 had been used?

Lane Tube

1 A DNA from crime scene cut with Enzyme 1 2 B DNA from crime scene cut with Enzyme 2 3 C DNA from Suspect 1 cut with Enzyme 1 4 D DNA from Suspect 1 cut with Enzyme 2 5 E DNA from Suspect 2 cut with Enzyme 1 6 F DNA from Suspect 2 cut with Enzyme 2

( - )

( + )

1 2 3 4 5 6

��




109Experiment

Inst

ruct

or’

s g

uid



study Questions and Answers

1. Define FLP’s and give their significance.

Fragment Length Polymorphisms (FLPs) are the variations in DNA sequences between individuals as determined by differences in restriction enzyme cleavage patterns (RFLPs) or differences in variable number of tandem repeats (VNTRs).

�. What is the most likely cause of Restriction Fragment Length Polymorphisms?

RFLPs are the result of variations in length of a given segment of genomic DNA be-tween two restriction endonuclease recognition sites among individuals of the same species.

�. What are Variable Number of Tandem Repeats (VNTRs)?

VNTRs are segments of DNA that contain sequences from 2 to 40 bases in length that repeat in tandem manner many times. These core units vary among individuals of the same species. The restriction sites are not altered. They are found in intergenic regions of DNA.

4. Who are the only individuals possessing the same DNA fingerprints?

Identical twins are the only individuals possessing the same DNA fingerprints.

5. List the steps involved in DNA fingerprinting from extraction of DNA through the matching of a suspect to a crime scene sample.

Steps involved in DNA Fingerprinting:

• Extraction of high molecular weight, DNA from blood, semen, skin, or hair roots, and purification.

• Restriction enzyme digest to produce fragments of various sizes. • Agarose gel electrophoresis to separate the fragments according to size. • Denaturation of DNA to separate the strands. • Southern Blot to transfer the results to a nylon membrane • Addition of a probe which will hybridize to the complementary DNA. • Identify the sizes of the DNA fragments that are hybridized to the probe. • Match the DNA fragment sizes of the suspects, with the DNA obtained from the

crime scene.

�. What type of human cells can be utilized for this technique?

DNA found in cells from blood, semen, skin, hair roots are utilized in DNA Finger-printing.

��

109Experiment


EVT 100202AM



A 0.8 % Agarose Gel Electrophoresis Reference Tables For DNA Staining with FlashBlue™ or InstaStain® Blue

B 0.8% Agarose Gel Electrophoresis Reference Tables For DNA Staining with InstaStain® Ethidium Bromide

C Quantity Preparations for Agarose Gel Electrophoresis

D Agarose Gel Preparation Step by Step Guidelines

E Staining and Visualization of DNA FlashBlue™ liquid

F Staining and Visualization of DNA InstaStain® Blue One-step Staining and destaining

G Staining and Visualization of DNA InstaStain® Blue Cards

H Staining and Visualization of DNA InstaStain® Ethidium Bromide Cards

Appendices

�4





Appendix


0.8% Agarose gel Electrophoresis Reference Tables(DNA staining with FlashBlue™ or Instastain® Blue)

Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C.1 for 0.8% agarose gels. The time for electrophoresis will vary from approximately 15 minutes to 2 hours depending upon various factors. Conduct the electrophoresis for the length of time determined by your instructor.

A

Amt ofAgarose

(g)


(ml)

Size of Gel(cm)

DistilledWater(ml)

TotalVolume

(ml)

7 x 7

7 x 10

7 x 14

0.23

0.39

0.46

0.6

1.0

1.2

29.4

49.0

58.8

+ =+



Table

A.1

30

50

60

Table

A.2

Amt ofAgarose

(g)

DilutedBuffer (1x)

(ml)

Size of Gel(cm)

7 x 7

7 x 10

7 x 14

0.23

0.39

0.46

30

50

60

+



For DNA analysis, the recommended electrophoresis buffer is Tris-acetate-EDTA, pH 7.8. The formula for diluting EDVOTEK (50x) concentrated buffer is one volume of buffer concen-trate to every 49 volumes of distilled or deionized water. Prepare buffer as required for your electrophoresis unit.

50x Conc.Buffer (ml)

DistilledWater (ml)+

EDVOTEKModel #

Total Volume Required (ml)

Electrophoresis (Chamber) Buffer

M6+

M12

M36

300

400

1000

Dilution

Table

B

6

8

20

294

392

980

Time and Voltage Guidelines(0.8% Gel)

Minimum / MaximumVolts

150

125

70

50

15 / 20 min

20 / 30 min

35 / 45 min

50 / 80 min

Table

C.1EDVOTEK Electrophoresis ModelM6+ M12 & M36

Minimum / Maximum

25 / 35 min

35 / 45 min

60 / 90 min

95 / 130 min



�5



109Experiment


Appendix


0.8% Agarose gel Electrophoresis Reference Tables(DNA staining with Instastain® Ethidium Bromide)



B

Amt ofAgarose

(g)


(ml)

Size of Gel(cm)

DistilledWater(ml)

TotalVolume

(ml)

7 x 7

7 x 10

7 x 14

0.15

0.23

0.31

0.4

0.6

0.8

19.6

29.4

39.2

+ =+


DNA Staining with InstaStain® Ethidium Bromide

Table

A.3

20

30

40

Table

A.4

Amt ofAgarose

(g)

DilutedBuffer (1x)

(ml)

Size of Gel(cm)

7 x 7

7 x 10

7 x 14

0.15

0.23

0.31

20

30

40

+


DNA Staining with InstaStain® Ethidium Bromide

Time and Voltage recommendations for EDVOTEK equipment are outlined in Table C.1 for 0.8% agarose gels. The time for electrophoresis will vary from approximately 15 minutes to 2 hours depending upon various factors. Conduct the electrophoresis for the length of time determined by your instructor.

For DNA analysis, the recommended electrophoresis buffer is Tris-acetate-EDTA, pH 7.8. The formula for diluting EDVOTEK (50x) concentrated buffer is one volume of buffer concen-trate to every 49 volumes of distilled or deionized water. Prepare buffer as required for your electrophoresis unit.

50x Conc.Buffer (ml)

DistilledWater (ml)+

EDVOTEKModel #

Total Volume Required (ml)

Electrophoresis (Chamber) Buffer

M6+

M12

M36

300

400

1000

Dilution

Table

B

6

8

20

294

392

980

Time and Voltage Guidelines(0.8% Gel)

Minimum / MaximumVolts

150

125

70

50

15 / 20 min

20 / 30 min

35 / 45 min

50 / 80 min

Table

C.1EDVOTEK Electrophoresis ModelM6+ M12 & M36

Minimum / Maximum

25 / 35 min

35 / 45 min

60 / 90 min

95 / 130 min

��





Appendix

C

To save time, the electrophoresis buffer and agarose gel solution can be prepared in larger quantities for sharing by the class. Unused diluted buffer can be used at a later time and solidified agarose gel solution can be remelted.

Bulk Electrophoresis Buffer

Quantity (bulk) preparation for 3 liters of 1x electro-phoresis buffer is outlined in Table D.

Batch Agarose gels (0.8%)

For quantity (batch) preparation of 0.8% agarose gels, see Table E.1.

1. Use a 500 ml flask to prepare the diluted gel buf-fer

2. Pour 3.0 grams of UltraSpec-Agarose™ into the prepared buffer. Swirl to disperse clumps.

3. With a marking pen, indicate the level of solution volume on the outside of the flask.

4. Heat the agarose solution as outlined previously for individual gel preparation. The heating time will require adjustment due to the larger total volume of gel buffer solution.

5. Cool the agarose solution to 60°C with swirling to promote even dis-sipation of heat. If evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step 3.

60˚C

Note: The UltraSpec-Agarose™ kit component is often labeled with the amount it contains. In many cases, the entire contents of the bottle is 3.0 grams. Please read the label carefully. If the amount of agarose is not specified or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount.


(ml)

DistilledWater(ml)

TotalVolume

(ml)

60 2,940 3000 (3 L)

=+

Bulk Preparation of Electrophoresis Buffer

Table

D

3.0 7.5 382.5 390

Batch Preparation of

0.8% UltraSpec-Agarose™

Amt ofAgarose

(g)

ConcentratedBuffer (50X)

(ml)+

DistilledWater(ml)

TotalVolume

(ml)=+

Table

E.1

6. Dispense the required volume of cooled agarose solution for casting each gel. The volume required is dependent upon the size of the gel bed and DNA staining method which will be used. Refer to Appendix A or B for guidelines.

7. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes. Then proceed with preparing the gel for electrophoresis.

Quantity Preparations for Agarose gel Electrophoresis

��



109Experiment


Appendix

EDVOTEK electrophoresis units include 7 x 7 cm or 7 x 14 cm gel casting trays.

A. Using Rubber dams:

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

B. Taping with labeling or masking tape:

• Extend 3/4 inch wide tape over the sides and bottom edge of the bed. • Fold the extended tape edges back onto the sides and bottom. Press contact

points firmly to form a good seal.

If gel trays and rubber end caps are new, they may be initially somewhat difficult to assemble. Here is a helpful hint:

2. Place a well-former template (comb) in the first set of notches at the end of the bed. Make sure the comb sits firmly and evenly across the bed.

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.

DAgarose gel Preparation - step by step guidelines

Place one of the black end caps with the wide “u” shaped slot fac-ing up on the lab bench.

Push one of the corners of the gel tray into one of the ends of the black cap. Press down on the tray at an angle, working from one end to the other until the end of the tray completely fits into the black cap. Repeat the process with the other end of the gel tray and the other black end cap.

Casting Agarose gels

3. Use a 250 ml flask or beaker to prepare the gel solution.

4. Refer to the appropriate Reference Table (i.e. 0.8%, 1.0% or 2.0%) for agarose gel preparation. Add the specified amount of agarose powder and buffer. Swirl the mixture to disperse clumps of agarose powder.

5. With a lab marking pen, indicate the level of the solution volume on the outside of the flask.

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

6. Heat the mixture to dissolve the agarose powder.

A. Microwave method:

• Cover the flask with plastic wrap to minimize evaporation.

• Heat the mixture on High for 1 minute. • Swirl the mixture and heat on High in bursts of 25 seconds

until all the agarose is completely dissolved.

B. Hot plate method:

• Cover the flask with aluminum foil to minimize evaporation. • Heat the mixture to boiling over a burner with occasional

swirling. Boil until all the agarose is completely dissolved.

Continue heating until the final solution appears clear (like water) with-out any undissolved particles. Check the solution carefully. If you see "crystal" particles, the agarose is not completely dissolved.

�8





Appendix

7. Cool the agarose solution to 60°C with careful swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume marked in step 5.

After the gel is cooled to �0°C:

• If you are using rubber dams, go to step 9. • If you are using tape, continue with step 8.

DO NOT POUR BOILING HOT AGAROSE INTO THE GEL BED.

Hot agarose solution may irreversibly warp the bed.

60˚C

+Black Red

Sample wells

–

During electrophoresis, the DNA samples migrate through the agarose gel towards the positive electrode.

Agarose gel Preparation step by step guidelines, continued D

8. Seal the interface of the gel bed and tape to prevent aga-rose solution from leaking.

• Use a transfer pipet to deposit a small amount of the cooled agarose to both inside ends of the bed.

• Wait approximately 1 minute for the agarose to solidify.

9. Place the bed on a level surface and pour the cooled agarose solution into the bed.

10. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes.

Preparing the gel for electrophoresis

11. After the gel is completely solidified, carefully and slowly remove the rubber dams or tape from the gel bed. Be especially careful not to damage or tear the gel wells when removing the rubber dams. A thin plastic knife, spatula or pipet tip can be inserted between the gel and the dams to break possible surface tension.

12. Remove the comb by slowly pulling 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 (refer to Table B on the Appendix page provided by your instructor).

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

�9



109Experiment


Appendix

Estaining and Visualization of DNA

FlashBlue™ Liquid stain

Preparation of FlashBlue™ stain from Concentrated solution

• Dilute 10 ml of 10x FlashBlue™ with 90 ml of distilled or deionized water in a flask. Mix well.

• Cover the flask and store it at room temperature until ready for gel staining.

Do not stain gel(s) in the electrophoresis apparatus.

staining and Destaining

1. Remove the agarose gel from its bed and and completely submerse the gel in a small, clean weighboat or lid from pipet tip rack containing

75 ml of 1x FlashBlue™ stain. Add additional stain if needed to com-pletely submerge the gel.

2. Stain the gel for 5 minutes.

Note: Staining the gel for longer than 5 minutes will necessitate an extended destaining time. Frequent changes of distilled water will expedite the process.

Wear Gloves and Goggles

5. Destain the gel for 20 minutes.

Dark blue bands will become visible against a light blue background. Additional destaining may yield optimal results.

6. Carefully remove the gel from the destaining liquid and examine the gel on a Visible Light Gel Visualization System. To optimize visibility, use the amber filter provided with EDVOTEK equipment.

3. Transfer the gel to another small tray and fill it with 250 - 300 ml of distilled water.

4. Gently agitate the tray every few minutes. Alternatively, place it on a shaking platform.

storage and Disposal of FlashBlue™ stain and gel

• Gels stained with FlashBlue™ 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.

• Stained gels which are not kept can be discarded in solid waste disposal. FlashBlue™ stain and destaining solutions can be disposed down the drain.

5 minutestaining

�0





Appendix

FOne-step staining and Destaining

with Instastain® Blue

1. Remove the 7 x 7 cm agarose gel from its bed and com-pletely submerse the gel in a small, clean tray containing

75 ml of distilled or deionized 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 containers


InstaStain™

One Step Stain and Destain

2. Wearing gloves, gently float a 7 x 7 cm card of InstaStain® Blue with the stain side (blue) facing the liquid.

Note: If staining a 7 x 14 cm gel, use two InstaStain® Blue cards.

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® 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.


Agarose gels can be stained and destained in one easy step with InstaStain™ Blue cards. This one-step method can be completed in approximately 3 hours, or can be left overnight.

�1



109Experiment


Appendix

1. After electrophoresis, place the agarose gel on a flat surface covered with plastic wrap.

2. Wearing gloves, place the blue dye side of the InstaStain® Blue card(s) on the gel.

3. Firmly run your fingers several times over the entire surface of the InstaStain® card to es-tablish good contact between the InstaStain® card and the gel.

InstaStain™

Patents Pending

DNA InstaStain™

Patents Pending

Patents Pending

InstaStain™

-----

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

staining and Visualization of DNAInstastain® Blue Cards

4. To ensure continuous contact between the gel and the InstaStain® card, place a gel casting tray and weight, such as a small empty beaker, on top of the InstaStain® card.

5. Allow the InstaStain® Blue to sit on the gel for 5 to 10 minutes.

6. After staining, remove the InstaStain® card.

If the color of the gel appears very light, wet the gel surface with buffer or distilled water and place the InstaStain® card 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 approximately 100 ml of distilled water to cover the gel.

9. Repeat destaining by changing the distilled water as needed.

Larger DNA bands will initially be visible as dark blue bands against a lighter blue background. When the gel is completely destained, larger DNA bands will become sharper and smaller bands will be visible. With additional de-staining, the entire background will become uniformly light blue. Destaining time may vary between 20 - 90 minutes.

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 filter provided with EDVOTEK equipment.

11. If the gel is too light and bands are difficult to see, repeat the staining and destaining procedures.


g

��





Appendix staining and Visualization of DNAInstastain® Blue Cards

continued

Destaining Notes for Instastain® Blue

• Use of 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 small-est 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® 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.

g

��



109Experiment


Appendix


1. After electrophoresis, place the gel on a piece of plastic wrap on a flat surface. Moisten the gel with a few drops of electrophoresis buffer.

2. Wearing gloves, remove the clear plastic pro-tective sheet, and place the unprinted side of the InstaStain® EtBr card(s) on the gel.

Disposal of Instastain

Disposal of InstaStain® cards and gels should follow institutional guidelines for chemical waste.

Additional Notes About staining

• If bands appear faint, or if you are not using EDVOTEK UltraSpec-Agarose™, gels may take longer to stain with InstaStain® EtBr. Repeat staining and increase the staining time an additional 10-15 minutes.

• DNA markers should be visible after staining even if other DNA samples are faint or absent. If markers are not visible, troubleshoot for problems with the electrophoretic separation.

Caution: Ethidium Bromide is a listed mutagen.

staining and Visualization of DNA Instastain® Ethidium Bromide Cards

3. Firmly run your fingers over the entire surface of the InstaStain® EtBr. Do this several times.

4. Place the gel casting tray and a small empty beaker on top to ensure that the InstaStain® card maintains direct contact with the gel surface.

Allow the InstaStain® EtBr card to stain the gel for 3-5 minutes.

5. After 10-15 minutes, remove the InstaStain® EtBr card. Transfer the gel to a ultraviolet (300 nm) transilluminator for viewing. Be sure to wear UV protective goggles.


h

1

2

3

4

5

Press firmly.

Moistenthe gel.

Place the InstaStain®card on the gel.

Place a small weight toensure good contact.

View on U.V. (300 nm) transilluminator

InstaStain™

Patents Pending

DNA InstaStain™

Patents Pending

Patents Pending

InstaStain™

-----

material safety Data sheetsFull-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.109

Experiment

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er

Ap

pea

ran

ce a

nd

Od

or

Sect

ion

IV -

Ph

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al/C

hem

ical

Ch

arac

teri

stic

sFl

ash

Po

int

(Met

ho

d U

sed

)

Exti

ng

uis

hin

g M

edia

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mab

le L

imit

sU

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L

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tin

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oin

t

Evap

ora

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n R

ate

(Bu

tyl A

ceta

te =

1)

Spec

ific

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vity

(H

0 =

1)

2

Ag

aro

se

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.

CA

S #9

012-

36-6

For

1% s

olu

tio

n 1

94 F

N

o d

ata

N

o d

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No

dat

a

No

dat

a

No

dat

a

Inso

lub

le -

co

ld

W

hit

e p

ow

der

, no

od

or

N.D

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o d

ata

No

dat

a

N

.D.

N.D

.

Wat

er s

pra

y, d

ry c

hem

ical

, car

bo

n d

ioxi

de,

hal

on

or

stan

dar

d f

oam

Poss

ible

fir

e h

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d w

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exp

ose

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flam

e

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ne

ED

VO

TE

K®

Stab

ility

Sect

ion

V -

Rea

ctiv

ity

Dat

aU

nst

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ion

VI -

Hea

lth

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ard

Dat

a

Inco

mp

atib

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Co

nd

itio

ns

to A

void

Ro

ute

(s)

of

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hal

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esti

on

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in?

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Stab

le

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ard

ou

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lym

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nM

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s to

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id

Will

No

t O

ccu

r

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lth

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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

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ho

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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

en. d

iluti

on

ven

tila

tio

n

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

Yes

Sp

lash

pro

of

go

gg

les

Imp

ervi

ou

s cl

oth

ing

to

pre

ven

t sk

in c

on

tact

No

neX

N

on

e

No

dat

a av

aila

ble

X

No

ne

Yes

Y

es

Yes

Inh

alat

ion

: N

o d

ata

avai

lab

le

In

ges

tio

n:

Larg

e am

ou

nts

may

cau

se d

iarr

hea

No

dat

a av

aila

ble

No

dat

a av

aila

ble

Trea

t sy

mp

tom

atic

ally

an

d s

up

po

rtiv

ely

Swee

p u

p a

nd

pla

ce in

su

itab

le c

on

tain

er f

or

dis

po

sal

No

rmal

so

lid w

aste

dis

po

sal

No

ne

No

ne

Ch

emic

al c

artr

idg

e re

spir

ato

r w

ith

fu

ll fa

cep

iece

.

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.

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r p

rote

ctiv

e eq

uip

men

t an

d S

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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

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

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material safety Data sheetsFull-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.

Stab

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Rea

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Dat

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Han

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


EVT 100202AM

EDVOTEK series 100 Electrophoresis Experiments:

Cat. # Title

101 Principles and Practice of Agarose Gel Electrophoresis

102 Restriction Enzyme Cleavage Patterns of DNA

103 PCR - Polymerase Chain Reaction

104 Size Determination of DNA Restriction Fragments

105 Mapping of Restriction Sites on Plasmid DNA

109 DNA Fingerprinting - Identification of DNA by Restriction Fragmentation Patterns

112 Analysis of Eco RI Cleavage Patterns of Lambda DNA

114 DNA Paternity Testing Simulation

115 Cancer Gene Detection

116 Sickle Cell Gene Detection (DNA-based)

117 Detection of Mad Cow Disease

118 Cholesterol Diagnostiics

124 DNA-based Screening for Smallpox

130 DNA Fingerprinting - Amplification of DNA for Fingerprinting

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