EDVO-Kit # 213 Cleavage of DNA with Restriction Enzymes · 213 Cleavage of DNA with Restriction Enzymes See page 3 for specific storage instructions. Some items require freezer storage

The Biotechnology Education Company ®

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

EVT 100217K

EDVO-Kit #

213Cleavage ofDNA with Restriction Enzymes

See page 3 for specific storage instructions. Some items require freezer storage.

ExpERimEnT OBjECTivE:

This experiment is an inquiry-based experiment to develop an understanding of DNA digestion by restriction enzymes and determining the size of DNA fragments by agarose gel electrophoresis.

2The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

213Experiment

Cleavage of DNA with Restriction Enzymes

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

Requirements 3

Background Information 4

Experiment Overview and General Instructions 7

Cleavage of DNA with Restriction Enzymes 8

Agarose Gel Electrophoresis 10

Size Determination of DNA Restriction Fragments 11

Experiment Analysis and Study Questions 13

Instructor’s Guide

Notes to the Instructor and Pre-Lab Preparations 15

Expected Results 18

Study Questions and Answers 19

Appendices 21

Material Safety Data Sheets 34

Table of Contents

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EVT 100217K

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

FAX: (301) 340-0582 • email: [email protected]


Contents Storage

A Eco RI Dryzyme™ endonuclease Room temp.B Bam HI Dryzyme™ endonuclease Room temp.C Restriction enzyme dilution buffer -20°C. D Restriction enzyme reaction buffer Room temp.E Water, qualified enzyme grade -20°C. F Supercoiled plasmid DNA 1 -20°C. G Supercoiled plasmid DNA 2 -20°C. H Lambda DNA -20°C. I Standard DNA fragments -20°C.

Store all of the following at Room temperature:

• 10x Gel Loading Solution • UltraSpec-Agarose™ powder • 50x concentrated electrophoresis buffer• InstaStain® Blue • FlashBlue™ liquid stain• 1 ml pipet• 100 ml plastic graduated cylinder• Microtest tubes with attached caps• Semi-log graph paper template

This experiment is designed for 6 groups.

Experiment Components

Requirements

• Horizontal Electrophoresis Apparatus• D.C. Power Supply• Automatic Micropipet (5-50 µl) & Disposable tips• Balance• Microwave Oven, Hot Plate, or Burner• White light visualization system*• Photodocumentation system (optional)• Waterbath• Pipet pumps or bulbs• 1 ml and 5 or 10 ml pipets• Disposable gloves and safety goggles• Hot gloves• Small plastic trays or large weigh boats• Lab Marking pens• Lab glassware - 20 & 250 ml beakers, 100 & 500 ml graduated cylinder• Metric rulers• Microtipped transfer pipets• Distilled or deionized water• Ice

* If performing alternate staining with InstaStain® Ethidium Bromide (must be purchased separately, Cat. # 2001), a UV Transil-luminator is required for DNA visualization.

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213Experiment


Background Information

Bgl I Bacillus globigii

Bam HI Bacillus amyloliquefaciens H

Eco RI Escherichia coli RY13

Eco RII Escherichia coli R 245

Hae III Haemophilus aegyptius

Hind III Haemophilus influenzae R4

Restriction Enzyme Organism

Sequence-specific, or Type II endonucleases, are commonly known as restriction enzymes. In contrast with nonspecific endonucleases, these enzymes generate reproducible fragments from DNA. They cleave double-stranded DNA by hy-drolyzing two phosphodiester bonds (one per strand) within defined nucleotide sequences. Over 3,000 restriction enzymes, which are extracted from a variety of bacterial strains, have been discovered since the first report by H.O. Smith and collaborators.

The discovery of restriction enzymes ushered in a new era of molecular genetics. These enzymes cut the DNA molecule in a highly specific and reproducible way. This, in turn, has lead to the development of molecular clon-ing and the mapping of genetic structures.

Restriction enzymes are endonucleases which catalyze the cleavage of the phosphodiester bonds within both strands of DNA. They require Mg+2 for activity and generate a 5 prime (5’) phosphate and a 3 prime (3’) hy-droxyl group at the point of cleavage. The distinguish-ing feature of restriction enzymes is that they only cut at very specific sequences of bases. Restriction enzymes are obtained from many different species of bacteria (including blue-green algae). To date, over 2,100 restric-tion enzymes have been discovered and catalogued.

The name of a restriction enzyme is derived from the genus and species of bac-terium from which it is isolated. The first letter of the genus name and first two letters of the species are combined to form the enzyme name. This is followed by a strain designation if applicable. In many instances, a bacterial strain con-tains more than one restriction endonuclease. When this occurs, each enzyme is assigned a Roman numeral. For example, Bam HI was the first enzyme activity reported from Bacillus amyloliquefaciens strain H (see figure 1).

A restriction enzyme requires a specific double stranded recognition sequence of nucleotides to cut DNA. Recognition sites are usually 4 to 8 base pairs in length. Cleavage occurs within or near the site. The cleavage positions are indicated by arrows. Recognition sites are frequently symmetrical, i.e., both DNA strands in the site have the same base sequence when read 5’ to 3’. Such sequences are called palindromes. Consider the recognition site and cleavage pattern of Eco RI as an example.

5'....G AATTC....3'3'....CTTAA G....5'

5'....GAATTC....3'3'....CTTAAG....5'

↑

↓

Eco RI

As shown above, Eco RI causes staggered cleavage of its site. The ends of the DNA fragments are called “sticky” or “cohesive” ends because the single-strand-ed regions of the ends are complementary.

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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 purpose without the written consent of EDVOTEK, Inc. Copyright © 2010 EDVOTEK, Inc., all rights reserved. EVT 100217K

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Some restriction enzymes, such as Hae III, introduce cuts that are opposite each other. This type of cleavage generates “blunt” ends.

The recognition sites of some restriction enzymes contain variable base positions. For example, Ava I recognizes:

5'....GG CC....3'3'....CC GG....5'

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

↑

↓

Hae III

(Py = pyrimidine = C or T and Pu = purine = G or A)

5'....CPyCGPuG....3'3'....GPuGCPyC....5'

↑

↓

Ava I

Keep in mind that A pairs with T and G pairs with C. Consequently, there are four possible sequences Ava I recognizes. Recognition sites of this type are called degenerate.

There are some recognition sites that are divided by a certain number of totally variable bases. For example, Bgl I recognizes:

There are 625 possible sequences Bgl I can cleave. The only bases the enzyme truly “recognizes” are the six G-C base pairs at the ends, which forms a palin-drome. In the case of Bgl I, these true recognition bases must always be separat-ed by 5 base pairs of DNA, otherwise the enzyme cannot properly interact with the DNA and cleave it. Recognition sites like that of Bgl I are called hyphenated sites.

In general, the longer the DNA molecule, the greater the probability that a given recognition site will occur. Therefore, human chromosomal DNA, which contains three billion base pairs, has many more recognition sites than a plasmid DNA containing only several thousand base pairs. However, very large DNA is difficult to isolate intact. During handling, it is randomly sheared to fragments in the range of 50,000 to 100,000 base pairs.

Plasmids and many viral DNAs are circular molecules. If circular DNA contains one recognition site for a restriction enzyme, then it will open up to form a lin-ear molecule when cleaved. By contrast, if a linear DNA molecule contains a sin-gle recognition site, when cleaved once it will generate two fragments. The size of the fragments produced depends on how far the sites are from each other. If a DNA molecule contains several recognition sites for a restriction enzyme, then under certain experimental conditions, it is possible that certain sites are cleaved and not others. These incompletely cleaved fragments of DNA are called partials.

(N = A, G, C or T)5'....GCCNNNNNGGC....3'3'....CGGNNNNNCCG....5'

↑

↓

Bgl I


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A B C

Complete Cleavage

Partial Cleavage

Recognition Sites

A

B

C

A

B

A B C

C

A

B C

A B C

B

Partials can arise if low amounts of enzyme are used or the reaction is stopped after a short time. In reality, reactions containing partials also contain some molecules that have been completely cleaved (figure 2).

Supercoiled plasmid DNA has a more compact and en-tangled shape than its corresponding non-supercoiled forms (linear, nicked and relaxed circles). Under the electrophoresis conditions used in the experiments you will be conducting, supercoiled DNA migrates faster than its linear form and linear DNA migrates faster than its nicked circular form. Catenanes migrate more slowly than single circles that are nicked during electrophoresis. Dimers migrate faster than trimers, which migrate faster than tetramers, etc. Catenanes give rise to the same final restriction enzyme cleavage patterns as their uncatenat-ed single forms.

Other forms of DNA, such as linear, circular or superhelical, are separated in the gel according to their charge, size and shape. One linear DNA example is Lambda DNA, a molecule from the E. coli bacteriophage lambda. It contains approximately 49,000 base pairs and has 5 recognition sites for Eco RI, and 7 for Hind III. The smaller fragments generated by a restriction enzyme, such as those generated by Hind III, may not be visible after separation on agarose gel electro-phoresis. Smaller fragments will be first to run off the gel during electrophoresis. There is less mass in the bands containing smaller fragments. They stain with less intensity and may be less detectable. Stoichiometric cleavage of a pure sample of DNA results in equimolar amounts of fragments.

Lambda phage DNA contains 10-16 base single-stranded regions at the 5’ and 3’ terminus which are self-complementary, called cos ends. To properly resolve lambda phage DNA fragments, they must be heated to 65°C before loading onto the gel. For example, the 4361 and 23130 base pair fragments will hybridize at the “cos” sites, and the amount of the 4361 base pair fragment will be decreased and hard to visualize on the stained gel.

This is an inquiry-based experiment where students will select to digest the three DNAs (two circular plasmid DNAs and a linear DNA) with Eco RI, Bam HI, or a mixture of the two enzymes and determine the size of the resulting fragments. Standard DNA fragments are provided to construct the standard curve.

Figure 1


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Experiment Overview and General Instructions

ExpERimEnT OBjECTivE:

This experiment is an inquiry-based experiment to develop an understanding of DNA digestion by restriction enzymes and determining the size of DNA fragments by agarose gel electrophoresis.

LABORATORY SAFETY

1. Gloves and goggles should be worn routinely as good laboratory prac-tice.

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.

• Although electrical current from the power source is automati-cally disrupted when the cover is removed from the electrophoresis apparatus, first turn off the power, then unplug the power source before disconnecting the leads and removing the cover.

• Turn off power and unplug the equipment when not in use.

5. EDVOTEK injection-molded electrophoresis units do not have glued junctions that can develop potential leaks. However, in the unlikely event that a leak develops in any electrophoresis apparatus you are using, IMMEDIATELY SHUT OFF POWER. Do not use the apparatus.

6. Always wash hands thoroughly with soap and water after handling reagents or bio-logical materials in the laboratory.

Wear Gloves and Goggles

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Each group should have the following materials

• Automatic micropipet & tips

• Microtest tubes

• Lab marker

Reagent or Biological

• Restriction Enzyme Reaction Buffer

• Plasmid DNA 1

• Plasmid DNA 2

• Lambda DNA

• Enzyme Grade Water

• Standard DNA Fragments

• Diluted Eco RI

• Diluted Bam HI

• 10x Gel Loading solution

Tube label

Rxn Buffer

DNA 1

DNA 2

DNA 3

Water

Markers

Eco RI

Bam HI

10x Gel Load

1. Plan an experiment using the table on the next page or construct a table similar to it.

2. Label 5 reaction tubes starting with "2".

The number "1" has been pre-assigned to Standard DNA markers which are ready for electrophoresis.

3. Add reaction buffer, DNA, water, and enzyme (always add the enzyme last) to reaction tubes you labeled. Use a FRESH micropipet tip for each transfer of DNA and enzyme.

4. Cap the reaction tubes and tap gently to mix. There should be no dense layer of enzyme solution at the bottom of the reaction tube.

5. Tap each tube on the lab bench or quick spin the tubes in a microcentrifuge to collect the contents at the bottom.

6. Incubate the tubes in a 37°C water bath for 30-60 minutes.

Note: Extending the restriction enzyme diges-tion to 60 minutes will ensure complete cleav-age of DNA.

7. After the 30-60 minute incubation is com-pleted, add 5 µl of 10x gel loading solution to reaction tubes to stop the reactions. Cap and mix by tapping. The reactions are ready for electrophoresis.

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Reminder

Do not cross-contaminate enzyme and DNA stocks by using the same pipet tip.

OptiOnal StOpping pOint After addition of 10x gel loading solution, which stops the reaction, samples are ready for electrophoresis. The samples may be stored in the refrigerator for electropho-resis at a later time.

Setting up DNA Digests (Select one or more digestions)

-------- Standard DNA Markers are ready to load for electrophoresis --------

DNA 1(5 µl)

ReactionTube

DNA 2(5 µl)

ReactionBuffer(5 µl)

FinalVolume(55 µl)

1

2

3

4

5

6

10xGel Load

(5 µl)

DNA 3(Lambda)

(5 µl)Eco RI(5 µl)

Bam HI(5 µl)

ReactionVolume(50 µl)

QualifiedWater

(to 50 µl)


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Agarose Gel Electrophoresis

Load the Samples

2. Before loading samples, equilibrate a 65°C waterbath or beaker of water for heating the tubes containing DNA frag-ments.

At 65°C, non-specific aggregation due to sticky ends generated by restriction enzyme digestions will melt. This will result in sharp individual DNA bands upon separation by agarose gel electrophoresis.

Run the Gel

5. After the DNA samples are loaded, set the power source at the required voltage and conduct electrophoresis for the length of time specified by your instructor.

6. After electrophoresis is completed, proceed to DNA staining and visualiza-tion (See Appendices for staining options and instructions).

7. Document and analyze the gel results.

Reminders:

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

+Black Red

Sample wells

–

After connecting the apparatus to the D.C. power source, check that current is flowing properly - you should see bubbles forming on the two electrodes.

3. Heat the samples, including the Standard DNA fragments for two minutes at 65°C. Allow the samples to cool for a few minutes.

4. Load 35 µl of each DNA sample in the following manner:

Lane Tube label 1 Markers Standard DNA Fragments 2 2 DNA Digest 1 3 3 DNA Digest 2 4 4 DNA Digest 3 5 5 DNA Digest 4 6 6 DNA Digest 5

Prepare the Gel

1. Prepare a 0.8% agarose gel for electrophoresis and DNA staining with Flash-Blue ™ or InstaStain® Blue. Refer to Appendix A. Alternatively, If DNA will be stained with InstaStain® Ethidium bromide, refer to Appendix B.

• Recommendedgelsize: 7x7cmor7x14cm • Numberofsamplewellsrequired: 6 • Placementofwell-formertemplate: firstsetofnotches • Agarosegelconcentrationrequired: 0.8%


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Size Determination of DNA Restriction Fragments

The sizes of DNA restriction fragments can be determined by migration distances after electrophoresis. The sizes of the "unknowns" (DNA digests in lanes 2-6) will be extrapolated by graphing their migration distances relative to the Standard DNA Fragments, for which the size of each fragment is known. The assignment of sizes for DNA fragments separated by agarose gel electrophoresis can have a ±10% margin of error.

DNA FRAGmENT SIzE DETERmINATION

1. Measure and record the distance traveled in the agarose gel by each Standard DNA fragment (except the largest 23,130 bp fragment, which will not fit in a straight line in step 4).

In each case, measure from the lower edge of the sample well to the lower edge of each band. Re-cord the distance traveled in centimeters (to the nearest millimeter).

2. Label the semi-log graph paper: • Label the non-logarithmic horizontal x-axis

"Migration Distance" in centimeters at equal intervals.

• Label the logarithmic vertical y-axis "Log base pairs". Choose your scales so that the data points are well spread out. Assume the first cycle on the y-axis represents 100-1,000 base pairs and the second cycle represents 1,000-10,000 base pairs.

3. For each Standard DNA fragment, plot the mea-sured migration distance on the x-axis versus its size in base pairs, on the y-axis.

4. Draw the best average straight line through all the points. The line should have roughly equal numbers of points scattered on each side of the line. Some points may be right on the line (see semi-log example at left).

Quick Reference:

Standard DNA fragment sizes - length is expressed in base pairs.

23130 9416 65574361300023222027 725 570

5. Measure the migration distance of each of the DNA fragments from the three restriction enzyme digestions (reactions 2, 3 and 4).

6. Using the graph of the Standard DNA fragments, determine the sizes in base pairs of each fragment from the restriction enzyme digestion reac-tions. Find the migration distance of a fragment on the x-axis - draw vertical line from that point until standard graph line is intersected. From the point of intersection, draw a second line horizontally to the y-axis and determine the approximate size of the fragment in base pairs (see semi-log example at left).

8

100,000

76

5

4

3

2

10,000

9

876

5

4

3

2

100

9

876

5

4

3

2

1,000

9

10,000 base pairs

1,000 base pairs

1 cm 2 cm 3 cm 4 cm 5 cm

Migration Distance(non-logarithmic x-axis)

Log

bas

e p

airs

(lo

gar

ith

mic

y-a

xis)

Example showing the plots of marker fragment migration distances on thenon-logarithmic x-axis versus its size, in base pairs,on the logarithmic y-axis

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80,000

100,000

70,000

60,000

50,000

40,000

30,000

20,000

90,000

800 700

600

500

400

300

200

100

9001,000

10,000

8,000 7,000

6,000

5,000

4,000

3,000

2,000

9,000

X-axis: Migration distance (cm)

1 cm 2 cm 3 cm 4 cm 5 cm 6 cm

Y-ax

is:

Bas

e Pa

irs

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Experiment Analysis & Study Questions

Observe and record the results of your experiment in your laboratory note-book or on a separate worksheet. Continue and answer the following study questions:

1. Can the size of a supercoiled plasmid DNA be determined by using stan-dard DNA fragments electrophoresed in parallel? Why?

2. An unknown DNA molecule was cleaved using several restriction en-zymes individually and in various combinations. The DNA fragment sizes were determined by agarose gel electrophoresis and the restriction enzyme recognition sites were mapped. Subsequently, the DNA was sequenced and an extra recognition site was found for one of the en-zymes. However, all the other data was consistent, within experimental errors, with sequence data. What are the simplest explanations for this discrepancy? Assume the DNA sequence had no errors.

3. Why are the Lambda DNA fragments heated prior to electrophoresis?

4. Predict the number of DNA fragments and their sizes if Lambda phage DNA were incubated and cleaved simultaneously with both Hind III and Eco RI (refer to the map below).

* The map is not drawn to scale. It serves to locate the relative sites of cleavage in base pairs.

1 21226 26104 31747 39168 44972 48502

A. Eco RI (5 Sites)

1 23130 27479 36895 37584 44141 48502 25157 37459

B. Hind III (7 Sites)

LAmBDA PHAGE DNA RESTRICTION ENzYmE mAP*48,502 Base Pairs


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instructor’s Guide

Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in planning and implementing this experiment with your students. These guidelines can be adapted to fit your specific 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 containing DNA markers run in parallel with other DNA samples. For DNA Sizing instructions, and other labora-tory extension 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

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Pre-Lab Preparations

Summary of Biologicalsand Reagents for each group

Restriction Enzyme Reaction Buffer

Plasmid DNA 1

Plasmid DNA 2

Lambda DNA

Enzyme Grade Water

Standard DNA Fragments

Diluted Eco RI

Diluted Bam HI

Quick Reference

D

F

G

H

E

I

A

B

Rxn Buffer

DNA 1

DNA 2

DNA 3

Water

Markers

Eco RI

Bam HI

30 µl

30 µl

30 µl

30 µl

40 µl

35 µl

30 µl on ice

30 µl on ice

pREpARATiOn OF dnA And mARkER SAmpLES

1. Thaw the following components on ice:

• Restriction enzyme dilution buffer (C) • Restriction enzyme reaction buffer (D) • Enzyme grade water (E) • Plasmid DNAs (F & G) • Lambda DNA (H) 2. Tap the tubes with your fingers

or on a table to get all contents to the bottom of the tube. Put them back on ice.

3. Label tubes and dispense the DNAs.

• 30 µl of plasmid DNA 1 to 6 microtest tubes labeled "DNA 1" • 30 µl of plasmid DNA 2 to 6 microtest tubes labeled

"DNA 2". • 30 µl of Lambda DNA to 6 microtest tubes labeled

"DNA 3"

4. Label 6 microtest tubes "Marker" and dispense 35 µl of the Stan-dard DNA Fragments (I) to each tube.

The experimental procedures consist of two major parts:

1) Restriction enzyme digestion of DNA 2) Agarose gel electrophoresis

Each group receives biologicals and reagents to plan and perform restriction en-zyme digestion reactions, which will be submitted to agarose gel electrophoresis. After electrophoresis, students analyze their gel and determine the locations of restriction enzyme cleavage sites on two circular plasmid DNAs.

AppROximATE TimE REQuiREmEnTS

• Prelab preparation and dispensing of biologicals and reagents take approxi-mately 1 hour.

• Allow 50-75 minutes for the restriction enzyme digestion and preparation of samples for electrophoresis. Extending the restriction enzyme digest incuba-tion time to 60 minutes will ensure complete cleavage of DNA.

• Approximate time for electrophoresis will vary from 40 minutes to 2 hours. See Agarose Gel Electrophoresis Hints and Help, Appendix I.

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Once reconstituted, the restriction enzymes are labile. Keep them cold and minimize handling. Prepare the enzymes no more than 30 minutes before use. Once diluted, enzymes must be used.

Do not cross-contaminate enzyme and DNA stocks by using the same pipet tip.

RECONSTITuTION OF DRYzYmE™ RESTRICTION ENzYmES

Students should perform the restriction enzyme digests within 30 minutes of reconstituting the Dryzymes™.

Prepare two Dryzymes™ Eco RI (A) and Bam HI (B) as specified in steps 5 - 10.

5. Make sure that the solid material is at the bottom of the tubes. If not, cen-trifuge the tubes in a microcentrifuge at full speed for 20 seconds or tap the tube on the lab bench.

6. Add 100 µl of Restriction Enzyme Dilution Buffer (C) to the solid at the bot-tom of each tube containing Dryzymes™ and allow the samples to hydrate for 1 minute.

7. Mix the samples vigorously by flicking the tubes with your finger or by vor-texing for 30 seconds until the solid appears to be completely dissolved.

8. Add 100 µl of Enzyme Grade Water (E) to each of the tubes of rehydrated Dryzymes™.

9. Mix or vortex the samples and centrifuge for 20 seconds or tap the tube on the lab bench.

10. Keep the reconstituted restriction enzyme on ice. Students should perform the restriction enzyme digests within 30 minutes.

ASSEmBLY OF STudEnT mATERiALS

11. The table at right summarizes the materials each student group will require for the restric-tion enzyme digestion part of this experiment.

Each group should have the following materials

• Automatic micropipet & tips

• Microtest tubes

• Lab marker

Reagent or Biological

• Restriction Enzyme Reaction Buffer

• Plasmid DNA 1

• Plasmid DNA 2

• Lambda DNA

• Enzyme Grade Water

• Standard DNA Fragments

• Diluted Eco RI

• Diluted Bam HI

• 10x Gel Loading solution

Tube label

Rxn Buffer

DNA 1

DNA 2

DNA 3

Water

Markers

Eco RI

Bam HI

10x Gel Load

Pre-Lab Preparations

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Results will vary based on the DNA and enzymes used. What follows is an example of a specific set of results from a digestion. The representative results show the relative positions of the DNA restriction fragments. Actual results will yield bands of varying sizes and intensities.

The migration rate of DNA fragments is inversely proportional to the log of their size in base pairs. However, the 23,130 base pair fragment is usually not included in the standard curve. Larger DNA fragments tend to migrate faster than one would predict. This is potentiated by increasing gel porosity and electric field strength. The percentage of agarose used for this experi-ment minimizes this effect.

Lane 1 Standard DNA Fragments (expressed in approximate base pairs)

23130 9416 6557 4361 3000 2322 2027 725 570 Lane 2 Plasmid - superhelical DNAs cannot be precisely measured using linear standard fragments.

Lane 3 Restriction 3710 bp ± 556 Enzyme 1 630 bp ± 95

Lane 4 Restriction 4340 bp ± 650 Enzyme 2

Lane 5 Restriction 2,080 bp ± 312 Enzyme 1 & 2 1630 bp ± 245 630 bp ± 95

( + )

( - )

1 2 3 4 5 6

23130 bp9416 bp

6557 bp4361 bp3000 bp2322 bp2027 bp

725 bp570 bp

Expected Results

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1. Can the size of a supercoiled plasmid DNA be determined by using stan-dard DNA fragments electrophoresed in parallel? Why?

No. The conformations of the standards and plasmid are different. Agarose gel electrophoresis separates DNA on the basis of its size and shape.

2. An unknown DNA molecule was cleaved using several restriction enzymes individually and in various combinations. The DNA fragment sizes were determined by agarose gel electrophoresis and the restric-tion enzyme recognition sites were mapped. Subsequently, the DNA was sequenced and an extra recognition site was found for one of the enzymes. However, all the other data was consistent, within experi-mental errors, with sequence data. What are the simplest explanations for this discrepancy? Assume the DNA sequence had no errors.

The extra site was very near another restriction site (example, 30 base pairs). A very small fragment will generally run off the gel in standard agarose gel electrophoresis procedures. In addition, the mass in the band containing the small fragment would probably be too low to de-tect by conventional staining methods. An alternate explanation to this observation is due to partial digestion. This particular recognition site may be more refractory to cleavage for a variety of reasons.

3. Why are the Lambda DNA fragments heated prior to electrophoresis?

Cos sequences will hybridize forming a large band, or fragments will not disassociate without heating.

continued

Study Questions & Answers

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1 21226 26104 31747 39168 44972 48502

A. Eco RI (5 Sites)

1 23130 27479 36895 37584 44141 48502 25157 37459

B. Hind III (7 Sites)


Fragment sizes: 21226 5148 4973 4268 3530 2027 1904 1584 1375 947 831 564 125

* The map is not drawn to scale. It serves to locate the relative sites of cleavage in base pairs.


Eco RI

double digest

Hind III

1 23130 27479 36895 37584 44141 48502 25157 37459

1 21226 26104 31747 39168 44972 48502

1 2 3 4 5 6 7 8 9 10 11 12 13

4. Predict the number of DNA fragments and their sizes if Lambda phage DNA were incubated and cleaved simultaneously with both Hind iii and Eco Ri (see map below).

If Lambda phage DNA were incubated and cleaved simultaneously with both Hind III and Eco RI, the result would be 13 bands - see diagram below:

Study Questions & answers

21

213Experiment #

EVT 100217K




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 Buffer and Agarose Quantity Preparations

D Agarose Gel Preparation

E Staining and Visualization of DNA - FlashBlue™

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

I Electrophoresis Hints and Help

Appendices

22


213Experiment


Appendix

A

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.

*0.77UltraSpec-Agarose™gelpercentageroundedupto0.8%

0.8% Agarose Gel Electrophoresis Reference Tables(DNA Staining with 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 approxi-mately 20 minutes to 2 hours depending upon various factors. Conduct the electro-phoresis for the length of time determined by your instructor.

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

Amt ofAgarose

(g)

ConcentratedBuffer (50x)

(ml)

Size of Gel(cm)

DistilledWater(ml)

TotalVolume

(ml)

7 x 7

7 x 14

0.23

0.46

0.6

1.2

29.4

58.8

+ =+

Individual 0.8%* UltraSpec-Agarose™ Gel

DNA Staining with FlashBlue™ or InstaStain® Blue

Table

A.1

30

60

Table

A.2

Amt ofAgarose

(g)

DilutedBuffer (1x)

(ml)

Size of Gel(cm)

7 x 7

7 x 14

0.23

0.46

30

60

+


DNA Staining with FlashBlue™ or InstaStain® Blue

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

50x Conc.Buffer (ml)

DistilledWater (ml)+

EDVOTEKModel #

Total Volume Required (ml)

Electrophoresis (Chamber) Buffer

M6+

M12

M36

Dilution

Table

B

300

400

1000

294

392

980

6

8

20

23


213Experiment


Appendix

If preparing a 0.8% gel with concentrated (50x) buffer, use Table A.3.

0.8% Agarose Gel Electrophoresis Reference Tables(DNA Staining with InstaStain® Ethidium Bromide)

If preparing a 0.8% gel with diluted (1x) buffer, use Table A.4.


B

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

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

Amt ofAgarose

(g)


(ml)

Size of Gel(cm)

DistilledWater(ml)

TotalVolume

(ml)

7 x 7

7 x 14

0.15

0.3

0.4

0.8

19.6

39.2

+ =+


DNA Staining with InstaStain® Ethidium Bromide

Table

A.3

20

40

Table

A.4

Amt ofAgarose

(g)

DilutedBuffer (1x)

(ml)

Size of Gel(cm)

7 x 7

7 x 14

0.15

0.3

20

40

+


DNA Staining with InstaStain® Ethidium Bromide

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

50x Conc.Buffer (ml)

DistilledWater (ml)+

EDVOTEKModel #

Total Volume Required (ml)

Electrophoresis (Chamber) Buffer

M6+

M12

M36

Dilution

Table

B

300

400

1000

294

392

980

6

8

20

24


213Experiment


Appendix

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

Buffer and Agarose Quantity Preparations

BuLk ELECTROphORESiS BuFFER

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


(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

60˚C


C

PREPARING AGAROSE GELS BY BATCH

For quantity (batch) preparation of 0.8% agarose gel solution, refer to Table E.1.

1. Use a 500 ml Pyrex flask or beaker to prepare the diluted gel buffer

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

3. With a marking pen, indicate the level of solu-tion volume on the outside of the flask.

4. Heat the agarose solution in the same manner as described for individual gel prepara-tion. 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 dissipation of heat. If evaporation has occurred, add distilled water to bring the solution up to the origi-nal volume as marked on the flask in step 3.

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.

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.

25


213Experiment


Appendix

d

EDVOTEK electrophoresis units include7x7cmor7x14cmgel 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 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.

Agarose Gel PreparationStep by Step Guidelines

Place one of the black end caps with the wide “u” shaped slot facing 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.

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.

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

26


213Experiment


Appendix

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

After the gel is cooled to 60°C:

•Ifyouareusingrubberdams,gotostep9. •Ifyouareusingtape,continuewithstep8.

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

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.

d

27


213Experiment


Appendix

E

Staining and Visualization of DNAFlashBlue™ Liquid Stain

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


Staining and destaining

1. Remove the agarose gel from its bed and completely submerse the gel in a small, clean tray containing 75 ml of 1x FlashBlue™ stain. Add addi-tional stain if needed to completely submerge the gel.

2. Stain the gel for no more than 5 minutes.

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.

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.

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

Storage and Disposal of 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.

28


213Experiment


Appendix Staining and Visualization of DNA

instaStain® BlueOne-step Staining and destaining

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.


InstaStain™

One Step Stain and Destain

1. Remove the 7 x 7 cm agarose gel from its bed and completely submerse the gel in a small, clean tray containing 75 ml of distilled or deionized water, or used electro-phoresis buffer. The agarose gel should be completely covered with liquid.

Examples of small trays include large weigh boats, or small plastic food containers

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

Note:Ifstaininga7x14cmgel,usetwoInstaStain®Bluecards.

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.


F

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.

29


213Experiment


Appendix

InstaStain™

Patents Pending

DNA InstaStain™

Patents Pending

Patents Pending

InstaStain™

- - - - -

1

2

3

4

5

6

Place gel on a flat surface covered with plastic wrap.

Place the InstaStain® card on the gel.

Place a small weight for approx. 5 minutes.

Transfer to a small tray for destaining.

Destain with 37°C distilled water.

Press firmly.

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 In-staStain® Blue card(s) on the gel.

(If staining a 7 x 14 cm gel, use two 7 x 7 cm cards)

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

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 dis-tilled 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 destaining, the entire background will become uniformly light blue.

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

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


GDo not stain gels in the

electrophoresis apparatus.

30


213Experiment


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 smallest tray available that will accommodate the gel. The gel should be completely submerged during destaining.

• Do not exceed 3 changes of water for destaining. Excessive destaining will cause the bands to be very light.

Storage and Disposal of InstaStain® 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 dis-posal.

• Destaining solutions can be disposed down the drain.

G

31


213Experiment


Appendix

DNA InstaStain™

Patents Pending

DNA InstaStain™

Patents Pending

- - - - -

- - - - -

1

2

3

4

5

Press firmly.

Moisten the gel.

Place the InstaStain® card on the gel.

Place a small weight to ensure good contact.

View on U.V. (300 nm) transilluminator


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. If staining a 7 x 14 cm gel, use two 7 x 7 cm In-staStain® EtBr cards. If staining a 7 x 7 cm gel, use one card.

Wearing gloves, remove the clear plastic protec-tive sheet from the InstaStain® EtBr card(s).

Place the unprinted side of the InstaStain® EtBr card(s) on the gel.

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

Visit our web site for an animated demonstration of InstaStain® EtBr.

Disposal of InstaStain

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

Caution: Ethidium Bromide is a listed mutagen.

Staining and Visualization of DNA instaStain® Ethidium Bromide Cards

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 in-crease the staining time an additional 10-15 minutes.

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 10-15 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 protec-tive goggles.


h

Wear glovesand UV Safety

goggles

• Markers (Standard DNA Fragments, DNA 100 bp ladder or DNA 200 bp ladder) should be visible after stain-ing even if the DNA samples are faint or absent. If markers are not visible, troubleshoot for problems with the electrophoretic separation.

32


213Experiment


Appendix

TO mAXImIzE SuCCESS:

1. The approximate time for electrophoresis will vary from experiment to experiment. A variety of factors, including gel concentration, will influ-ence electrophoresis time. Generally, the higher the voltage applied, the faster the samples mi-grate. However, depending upon the apparatus configuration and the distance between the two electrodes, individual electrophoresis units will separate DNA at different rates.

2. Do not move the apparatus after the samples have been loaded.

• Moving the apparatus will dislodge the samples from the wells into the buffer and will compromise results.

• If it is absolutely necessary to move the apparatus during electrophoresis, you may safely do so after the tracking dye has mi-grated at least 1 cm from the wells into the gel.

3. For optimal DNA fragment separation, do not use voltages higher than 125 volts for agarose gel electrophoresis. Higher voltages can over-heat and melt the gel.

4. The DNA samples contain tracking dye, which moves through the gel ahead of most DNA (ex-cept extremely small fragments). Migration of the tracking dye will become clearly visible in the gel after approximately 10-15 minutes.

( + )

( - )

TRACKINGDYE

Migration3.5 - 4.0 cm

Samplewells

Agarose Gel Electrophoresis Hints and Help

i

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

5. If DNA fragments are similar in size, fragments will migrate close to one another.

• In general, longer electrophoretic runs will increase the separation between fragments of similar size.

• Experiments which involve measurement of fragment molecular size or weight should be run at the recommended optimal time to ensure adequate separation.

6. Electrophoresis should be terminated when the tracking dye has moved a minimum of 3.5 to 4 centimeters from the wells for 7 x 7 cm gels, or 5-8 centimeters for 7 x 14 cm gels. Terminate the electrophoresis before the tracking dye moves off the end of the gel.

• For optimal results, stain the gel immedi-ately after electrophoresis.

• For convenience, the power source can be connected to a household automatic light timer to terminate the electrophoretic sepa-ration and avoid running samples off the end of the gel.

Figure is not drawn to scale.

33


213Experiment


AppendixAgarose Gel Electrophoresis Hints and Help

TO AvOid COmmOn pROBLEmS:

To avoid potential problems, some suggestions and reminders are listed below.

7. Use only distilled or deionized water to prepare buffers and gels. Do not use tap water.

8. To ensure that DNA bands are well resolved, make sure the gel formulation is correct and that electrophoresis is conducted for the recommend-ed optimal amount of time.

9. Correctly dilute the concentrated buffer for preparation of both the gel and electrophore-sis (chamber) buffer. Remember that without buffer in the gel, there will be no DNA mobil-ity. Check that the gel is completely submerged under buffer during electrophoresis.

10. For optimal results, use fresh electrophoresis buf-fer prepared according to instructions.

11. Before performing the actual experiment, prac-tice sample delivery techniques to avoid diluting the sample with buffer during gel loading.

12. To avoid loss of DNA fragments into the buffer, make sure the gel is properly oriented so the samples are electrophoresed from the negative electrode (cathode) towards the positive elec-trode (anode).

13. To avoid obtaining gel results that are missing small DNA fragments (small fragments move faster), remember that the tracking dye in the sample moves through the gel ahead of the smallest DNA fragments. Terminate the electro-phoresis before the tracking dye moves off the end of the gel.

14. If DNA bands appear faint after staining and destaining, repeat the procedure. Staining for a longer period of time will not harm the gel. Re-stained gels may require longer destaining.

CARE And mAinTEnAnCE OF ThE ELECTROphORESiS AppARATuS

15. The temperature of the melted agarose which is poured into the bed during gel casting should not exceed 60°C. Hot agarose solution may ir-reversibly warp the casting tray.

16. Avoid touching the fragile platinum electrodes.

17. Power should always be turned off and leads disconnected from the power source when the cover is removed from the apparatus.

18. To clean the apparatus chamber, gel casting tray and combs, rinse thoroughly with tap water. Give the items a final rinse with distilled water. Let them air dry. Do not use detergents of any kind, or expose the apparatus to alcohols.

19. EDVOTEK injection-molded electrophoresis units do not have glued junctions that can develop potential leaks. In the unlikely event that a leak develops in any electrophoresis apparatus you are using, IMMEDIATELY SHUT OFF POWER. Do not use the apparatus.

i

Order Online

Visit EDVOTEK’sweb site for the mostcomprehensive line of experiments for biotechnology & biology education.

material Safety Data SheetsFull-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.213

Experiment

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h O

SHA

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om

mu

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nSt

and

ard

. 29

CFR

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0.12

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tan

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TITY

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Nam

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Dis

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N

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No

ne

No

ne

Mat

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l Saf

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Dat

a Sh

eet

May

be

use

d t

o c

om

ply

wit

h O

SHA

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d C

om

mu

nic

atio

nSt

and

ard

. 29

CFR

191

0.12

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tan

dar

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ific

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ts.

IDEN

TITY

(A

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sed

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Lab

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lan

k sp

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no

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ny

item

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form

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n is

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spac

e m

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arke

d t

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dic

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that

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Sect

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IM

anu

fact

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r's

Nam

e

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II -

Haz

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gre

die

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Info

rmat

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Emer

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r in

form

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Dat

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Sig

nat

ure

of

Prep

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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

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veR

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ville

, MD

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50

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dlin

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Step

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ater

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PER

EAC

47.

3042

0.82

No

dat

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reg

ula

tio

ns.

Kee

p t

igh

tly

clo

sed

. St

ore

in c

oo

l, d

ry p

lace

No

ne

MIO

SH/O

SHA

ap

pro

ved

, SC

BA

Req

uir

ed

Ru

bb

erC

hem

. saf

ety

go

gg

les

Ru

bb

er b

oo

ts

35

213Experiment

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

Stab

ility

Sect

ion

V -

Rea

ctiv

ity

Dat

aU

nst

able

Sect

ion

VI -

Hea

lth

Haz

ard

Dat

a

Inco

mp

atib

ility

Co

nd

itio

ns

to A

void

Ro

ute

(s)

of

Entr

y:In

hal

atio

n?

Ing

esti

on

?Sk

in?

Oth

er

Stab

le

Haz

ard

ou

s Po

lym

eriz

atio

nM

ay O

ccu

rC

on

dit

ion

s to

Avo

id

Will

No

t O

ccu

r

Hea

lth

Haz

ard

s (A

cute

an

d C

hro

nic

)

Car

cin

og

enic

ity:

NTP

?O

SHA

Reg

ula

tio

n?

IAR

C M

on

og

rap

hs?

Sig

ns

and

Sym

pto

ms

of

Exp

osu

re

Med

ical

Co

nd

itio

ns

Gen

eral

ly A

gg

rava

ted

by

Exp

osu

re

Emer

gen

cy F

irst

Aid

Pro

ced

ure

s

Sect

ion

VII

- Pr

ecau

tio

ns

for

Safe

Han

dlin

g a

nd

Use

Step

s to

be

Take

n in

cas

e M

ater

ial i

s R

elea

sed

fo

r Sp

illed

Was

te D

isp

osa

l Met

ho

d

Prec

auti

on

s to

be

Take

n in

Han

dlin

g a

nd

Sto

rin

g

Oth

er P

reca

uti

on

s

Sect

ion

VIII

- C

on

tro

l Mea

sure

s

Ven

tila

tio

nLo

cal E

xhau

stSp

ecia

l

Mec

han

ical

(G

ener

al)

Res

pir

ato

ry P

rote

ctio

n (

Spec

ify

Typ

e)

Pro

tect

ive

Glo

ves

Oth

er P

rote

ctiv

e C

loth

ing

or

Equ

ipm

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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,

nit

rog

en o

xid

es, h

ydro

gen

bro

mid

e g

as

X

N

on

e

Yes

Y

es

Yes

No

dat

a av

aila

ble

Irri

tati

on

to

mu

cou

s m

emb

ran

es a

nd

up

per

res

pir

ato

ry t

ract

No

dat

a

Trea

t sy

mp

tom

atic

ally

an

d s

up

po

rtiv

ely

Wea

r SC

BA

, ru

bb

er b

oo

ts, r

ub

ber

glo

ves

Mix

mat

eria

l wit

h c

om

bu

stib

le s

olv

ent

and

bu

rn in

a c

hem

ical

inci

ner

ato

r eq

uip

ped

aft

erb

urn

er a

nd

scr

ub

ber

Use

in c

hem

ical

fu

me

ho

od

wit

h p

rop

er p

rote

ctiv

e la

b g

ear.

Mu

tag

en

Yes

Ch

em. f

um

e h

oo

d

No

N

on

e

Ru

bb

er

C

hem

. saf

ety

go

gg

les

R

ub

ber

bo

ots

Use

in c

hem

ical

fu

me

ho

od

wit

h p

rop

er p

rote

ctiv

e la

b g

ear.

Acu

te: M

ater

ial i

rrit

atin

g t

o m

uco

us

mem

bra

nes

, up

per

res

pir

ato

ry t

ract

, eye

s, s

kin

Ch

ron

ic:

May

alt

er g

enet

ic m

ater

ial

SCB

A

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

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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

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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

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ne

Nu

mb

er

Tele

ph

on

e N

um

ber

fo

r in

form

atio

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Dat

e Pr

epar

ed

Sig

nat

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of

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pti

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MD

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mo

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s)]

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Bo

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Un

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D

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CA

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Stab

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V -

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Co

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cute

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cin

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ye c

on

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ay c

ause

irri

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on

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o d

ata

avai

lab

le f

or

oth

er r

ou

tes.

No

dat

a av

aila

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cau

se s

kin

or

eye

irri

tati

on

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ne

rep

ort

ed

Trea

t sy

mp

tom

atic

ally

an

d s

up

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se c

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wit

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s am

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of

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r ey

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sp

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nd

loca

l reg

ula

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ns.

Avo

id e

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nd

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n c

on

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ne

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No

ne

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ne

Yes

Spla

sh p

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f g

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gle

s

No

ne

req

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ed

Avo

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nd

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n c

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Mat

eria

l Saf

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Dat

a Sh

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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

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or

spec

ific

req

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ts.

IDEN

TITY

(A

s U

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on

Lab

el a

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t)N

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lan

k sp

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are

no

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If a

ny

item

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ot

app

licab

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o in

form

atio

n is

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spac

e m

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e m

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dic

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that

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form

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Dat

e Pr

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Sig

nat

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pti

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Ad

dre

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Nu

mb

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Sta

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Co

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EDV

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1467

6 R

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Ch

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Un

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Stab

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ard

s (A

cute

an

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Car

cin

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Reg

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tio

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ns

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Exp

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Gen

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gg

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Exp

osu

re

Emer

gen

cy F

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Aid

Pro

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ure

s

Sect

ion

VII

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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

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on

s to

be

Take

n in

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dlin

g a

nd

Sto

rin

g

Oth

er P

reca

uti

on

s

Sect

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on

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l Mea

sure

s

Ven

tila

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nLo

cal E

xhau

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l

Mec

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(G

ener

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Res

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Spec

ify

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e)

Pro

tect

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Glo

ves

Oth

er P

rote

ctiv

e C

loth

ing

or

Equ

ipm

ent

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rk/H

ygie

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es

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Pro

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ion

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ard

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s D

eco

mp

osi

tio

n o

r B

ypro

du

cts

X X

Trea

t sy

mp

tom

atic

ally

an

d s

up

po

rtiv

ely

Rin

se c

on

tact

ed a

rea

wit

h c

op

iou

s am

ou

nts

of

wat

er.

No

ne

req

uir

ed

No

ne

Yes

Y

es

Y

es

May

cau

se s

kin

or

eye

irri

tati

on

No

ne

rep

ort

ed

Rin

se c

on

tact

ed a

rea

wit

h c

op

iou

s am

ou

nts

of

wat

er.

Ob

serv

e al

l fed

eral

, sta

te, a

nd

loca

l reg

ula

tio

ns.

Avo

id e

ye a

nd

ski

n c

on

tact

.

No

ne

Ch

emic

al c

artr

idg

e re

spir

ato

r w

ith

org

anic

vap

or

cart

rid

ge.

Yes

Yes

Yes

No

ne

yes

Sp

lash

pro

of

go

gg

les

Do

no

t in

ges

t. A

void

co

nta

ct w

ith

ski

n, e

yes

and

clo

thin

g.

Was

h t

ho

rou

gh

ly a

fter

han

dlin

g.

No

ne

No

ne

kno

wn

Sulf

ur

oxi

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213Experiment


Convenient, Time-SavingSeries 100 Electrophoresis Experiments from EDVOTEK:

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

Visit our web site for information about the above experiments and other products

in EDVOTEK’s comprehensive offerings for biotechnology and biology education.

Order Online

Documents

EDVO-Kit # 213 Cleavage of DNA with Restriction Enzymes · 213 Cleavage of DNA with Restriction Enzymes See page 3 for specific storage instructions. Some items require freezer storage