ap® investigation #9 bio #9 v3.pdf...ward’s to adapt to the specifications outlined in ap biology...

AP® InvestIgAtIon #9 Genetics and information transfer: restriction enzyme – teacher’s Guide

©2012, Ward’s Natural Science

All Rights Reserved, Printed in the U.S.A.

US: www.Ward’sci.com

Canada: www.Ward’sci.ca

Call “Us” at 1.800.955.2660 for Technical Assistance

250-7458 v.5/12

Kit # 36-7409

abstract 1

General overview 2

recordinG data 3

material requirements/checklist 5

curriculum aliGnment 6

learninG objectives 6

time requirements 6

safety Precautions 7

Pre-lab PreParation 9

student Guide contents

backGround 12

safety 17

Part 1: modelinG dna and restriction diGests 18(structured inquiry)

Part 2: aGarose Gel electroPhoresis 25(Guided inquiry)

assessment questions/answer keys 32

Part 3: desiGn an exPeriment 37(oPen inquiry)

restriction enzyme cards 39

material safety data sheets 50

table of Contents

**AP® and the Advanced Placement Program are registered trademarks of the College Entrance Examination Board. The activity and materials in this kit were developed and prepared by WARD’S Natural Science Establishment, which bears sole responsibility for their contents..





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Genetics and information transfer: restriction enzyme – teacher’s Guide Kit # 36-7409

AbstrACtThis lab starts with a scenario to illustrate how students might use agarose gel electrophoresis technology to solve a problem. The students will use hands-on modeling of complex molecular processes to understand the necessary steps and underlying logic in common biotechnology methods. After modeling the processes, the students will compare the DNA samples found at a crime scene to DNA isolated from fictional “suspects” using hands-on gel electrophoresis. The students will compare fragments of known size to fragments of an unknown size to calculate relative molecular weights, and match fragment patterns from unknown samples to known samples. Students are encouraged to consider the benefits, risks and ethics that come with the availability of such powerful techniques.





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generAl overvIewThe College Board has revised the AP Biology curriculum to begin implementation in the fall of 2012. Advanced Placement (AP) is a registered trademark of the College Entrance Examination Board. The revisions were designed to reduce the range of topics covered, to allow more depth of study and increased conceptual understanding for students. There is a shift in laboratory emphasis from instructor-designed demonstrations to student-designed investigations. While students may be introduced to concepts and methods as before, it is expected that they will develop more independent inquiry skills. Lab investigations now incorporate more student-questioning and experimental design. To accomplish this, the College Board has decreased the minimum number of required labs from 12 to 8 while keeping the same time requirement (25% of instruction time devoted to laboratory study). The College Board has defined seven science practices that students must learn to apply over the course of laboratory study. In brief, students must:

1. Use models

2. Use mathematics (quantitative skills)

3. Formulate questions

4. Plan and execute data collection strategies

5. Analyze and evaluate data

6. Explain results

7. Generalize data across domains

The College Board published 13 recommended laboratories in the spring of 2011. They can be found at: http://advancesinap.collegeboard.org/science/biology/lab

Many of these laboratories are extensions of those described in the 12 classic labs that the College Board has used in the past. The materials provided in this lab activity have been prepared by Ward’s to adapt to the specifications outlined in AP Biology Investigative Labs: An Inquiry-Based Approach (2012, The College Board). Ward’s has provided instructions and materials in the AP Biology Investigation series that complement the descriptions in this College Board publication. We recommend that all teachers review the College Board material as well as the instructions here to get the best understanding of what the learning goals are. Ward’s has structured each new AP investigation to have at least three parts: Structured, Guided, and Open Inquiry. Depending on a teacher’s syllabus, s/he may choose to do all or only parts of the investigations in scheduled lab periods.

The College Board requires that a syllabus describe how students communicate their experimental designs and results. It is up to the teacher to define how this requirement will be met. Having students keep a laboratory notebook is one straightforward way to do this.





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reCordIng dAtA In A lAborAtory notebook

All of the Ward’s Investigations assume that students will keep a laboratory notebook for student-directed investigations. A brief outline of recommended practices to set up a notebook, and one possible format, are provided below.

1. A composition book with bound pages is highly recommended. These can be found in most stationary stores. Ward’s offers several options with pre-numbered pages (for instance, item numbers 32-8040 and 15-8332). This prevents pages from being lost or mixed up over the course of an experiment.

2. The title page should contain, at the minimum, the student’s name. Pages should be numbered in succession.

3. After the title page, two to six pages should be reserved for a table of contents to be updated as experiments are done so they are easily found.

4. All entries should be made in permanent ink. Mistakes should be crossed out with a single line and should be initialed and dated. This clearly documents the actual sequence of events and methods of calculation. When in doubt, over-explain. In research labs, clear documentation may be required to audit and repeat results or obtain a patent.

5. It is good practice to permanently adhere a laboratory safety contract to the front cover of the notebook as a constant reminder to be safe.

6. It is professional lab practice to sign and date the bottom of every page. The instructor or lab partner can also sign and date as a witness to the veracity of the recording.

7. Any photos, data print-outs, or other type of documentation should be firmly adhered in the notebook. It is professional practice to draw a line from the notebook page over the inserted material to indicate that there has been no tampering with the records.

For student-directed investigations, it is expected that the student will provide an experimental plan for the teacher to approve before beginning any experiment. The general plan format follows that of writing a grant to fund a research project.

1. Define the question or testable hypothesis.

2. Describe the background information. Include previous experiments.

3. Describe the experimental design with controls, variables, and observations.

4. Describe the possible results and how they would be interpreted.

5. List the materials and methods to be used.

6. Note potential safety issues.

(continued on next page)





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reCordIng dAtA In A lAborAtory notebook (continued)

After the plan is approved: 7. The step-by-step procedure should be documented in the lab notebook. This includes recording

the calculations of concentrations, etc., as well as the weights and volumes used.

8. The results should be recorded (including drawings, photos, data print outs, etc.).

9. The analysis of results should be recorded.

10. Draw conclusions based on how the results compared to the predictions.

11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance, and uncontrolled variables.

12. Further study direction should be considered.

The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following questions, which also might arise in peer review:

• Explain the purpose of a procedural step.

• Identify the independent variables and the dependent variables in an experiment.

• What results would you expect to see in the control group? The experimental group?

• How does XXXX concept account for YYYY findings?

• Describe a method to determine XXXX.





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MAterIAls ProvIded In kIt MAterIAls needed bUt not ProvIded

Units per kit

DescriptionElectrophoresis chambers

£ 1 Coupon, Perishable Material* Aluminum foil

£ 200 Clear connectors Graduated cylinder 1 L

£ 1 Prepared agarose, 0.8%, 200 mL Miicrofuge tube racks

£ 32 Microfuge tubes, 1.5 mL Hot plate

£ 8 Agarose gel staining tray Gel casting trays with 16 well combs

£ 1 Tris-Borate-Edta, 5 x 500 mL Power supplies

£ 1 Ward’s QUIKView DNA Stain, Hot water bath or microwave

£ 6 Zip plastic bags 10 µL micropipets

£ 20 Blue pop bead, 10 mm (Set/5) Metric ruler

£ 20 Yellow pop bead, 10 mm (Set/5) Calculators

£ 20 Orange pop bead, 10 mm (Set/5) Safety goggles

£ 20 Green Pop Bead 10 mm (Set/5) Chemical resistant lab aprons

£ 1 Felt Tip Marker, Orange Chemical resistant gloves

£ 1 Felt Tip Marker, Blue Distilled water

£ 1 Felt Tip Marker, YellowHeat resistant gloves/mitts

£ 1 Felt Tip Marker, Green oPtIonAl MAterIAls (not ProvIded)

£ 1 Instructions (this document) White light box

Lambda DNA (uncut)

Restriction enzymes: BamHI, EcoRI, HindIII, etc.

MAterIAls CheCklIst

Call “Us” at 1.800.955.2660 for

Technical Assistance Visit “Us” on-line at

www.Ward’sci.com

for U.S. Customers

www.Ward’sci.ca

for Canadian Customers

or

* - It is recommended that you redeem your coupon for live/

perishable materials as soon as possible and specify your preferred delivery date. Generally, for timely delivery, at least a week’s advance

notice is preferred.





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CUrrICUlUM AlIgnMentBig Ideas

Big Idea 3: Living systems store, retrieve, transmit, and respond to information essential to life processes.

Big Idea 1: The process of evolution drives the diversity and unity of life.

Enduring Understandings

C3: Populations of organisms continue to evolve.

3A1: DNA, and in some cases RNA, is the primary source of heritable information.

3A3: The chromosomal basis of inheritance provides an understanding of the pattern of passage (transmission) of genes from parent to offspring.

Science Practices

3.1 The student can pose scientific questions.

6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.

leArnIng objeCtIvesThe student can justify the claim that humans can manipulate heritable information by identifying at least two commonly used technologies (3A1 & SP 6.4).

The student is able to pose questions about ethical, social, or medical issues surrounding human genetic disorders [an application of genetic engineering] (3A3 & SP 3.1).

tIMe reqUIreMents

Part 1: Modeling DNA and Restriction Digests (Structured Inquiry)

45 minutes total: (Steps 1 & 2 can be done as homework; Steps 3-6 can be done in-class, approx. 15 min.)

Part 2: Electrophoresis Techniques and Analysis (Guided Inquiry)

60 minutes (assuming teacher pre-pours gel), plus analysis

Part 3: Open InquiryVaries, depending on students’ experimental designs.

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Kit # 36-7409

This lab activity is aligned with the 2012 AP Biology Curriculum (registered trademark of the College Board). Listed below are the aligned Content Areas (Big Ideas and Enduring Understandings), the Science Practices, and the Learning Objectives of the lab as described in AP Biology Investigative Labs: An Inquiry Approach (2012). This is a publication of the College Board that can be found at http://advancesinap.collegeboard.org/science/biology/lab.





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sAfety PreCAUtIons Lab-Specific Electrical Safety

CAUTION: The power supply produces a high voltage that can cause severe electrical shock if handled improperly. For safe operation, follow all directions and precautions.

Before using the electrophoresis apparatus:

Examine all components of the electrophoresis apparatus prior to each use, including all cords, plugs, jacks, the electrophoresis chamber itself, and the power supply.

Do not operate the electrophoresis apparatus in a damp or humid environment. Any condensed moisture may short out electrical components.

You may wish to designate one area of the laboratory specifically for electrophoresis equipment, where cells and power supplies are connected. Ensure that power cords and patch cords are free from moisture, and that any wall outlet is properly wired; i.e., that correct polarity exists (use a circuit tester).

Be sure that students are well acquainted with the correct procedure for making electrical connections. Students should be supervised at all times when performing this investigation.

Do not come in personal contact with or allow metal or any conductive material to come in contact with the reservoir buffer or the electrophoretic cell while the power supply is on.

Lab-Specific Chemical Safety

Tris-Borate-EDTA is slightly toxic if ingested.

Ward’s QuickView DNA stain will stain clothing and skin.

General Safety

The teacher should be familiar with safety practices and regulations in their school (district and state). The teacher should know what needs to be treated as hazardous waste and how to properly dispose of non-hazardous chemicals or biological material.

Consider establishing a safety contract that students and their parents must read and sign off on. This is a good way to identify students with allergies to things like latex so that you (and they) will be reminded of what particular things may be risks to individuals. A good practice is to include a copy of this contract in the student lab book (glued to the inside cover).

Students should know where all emergency equipment (safety shower, eyewash station, fire extinguisher, fire blanket, first aid kit etc.) is located.

Make sure students remove all dangling jewelry and tie back long hair before they begin.

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Remind students to read all instructions, Material Safety Data Sheets (MSDSs) and live care sheets before starting the lab activities and to ask questions about safety and safe laboratory procedures. Appropriate MSDSs can be found on the last pages of this booklet. Additionally, the most updated versions of these resources can be found atwww.Ward’sci.com, under Living Materials http://Ward’sci.com/article.asp?ai=1346. (Note that in this particular lab, there are no live materials that require a live care sheet.

In student directed investigations, make sure that collecting safety information (like MSDSs) is part of the experimental proposal.

As general laboratory practice, it is recommended that students wear proper protective equipment, such as gloves, safety goggles, and a lab apron.

At end of lab:

All laboratory bench tops should be wiped down with a 20% bleach solution or disinfectant to ensure cleanliness.

Remind students to wash their hands thoroughly with soap and water before leaving the laboratory.

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sAfety PreCAUtIons General Safety (continued)





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Pre-lAborAtory PrePArAtIonPart 1: Structured Inquiry Lab Prep

1. At the end of this booklet, find the master pages for restriction enzyme cards, alkali cards, and probe cards, as well as the DNA sequences for the characters in the scenario.

2. Using scissors, cut the pages into DNA sequence strips or the individual cards. Each group performing the exercise will require one of each card and a DNA strip.

TIP: Have at least two groups get murderer strips.

3. Using the markers included in the kit and the key below, color in the bases in the restriction enzyme cards and the probe cards.

A – yellow G – green

C – blue T – orange

OPTIONAL: Have students assist you with this step.

Part 2: Guided Inquiry Lab Prep

1. Prepare 1X TBE Running Buffer

Add 160 mL of distilled water to 40 mL of 5X TBE concentrate to obtain 200 mL of 1X buffer.

NOTE: Any remaining 1X TBE buffer may be stored at room temperature in a clean container. Once diluted, the buffer will provide best results if used within 18 months. Any remaining 5X TBE buffer concentrate may be stored in the original container. If, over time, the concentrated buffer develops a white precipitate, it should be discarded.

2. Melt Agarose

NOTE: The bottle of prepared agarose provides enough material to produce at least thirteen 15 mL gels.

NOTE: Always wear heat-protective gloves when handling a hot bottle of agarose.

Melt the agarose using either a hot waterbath or microwave.

Water bath: Loosen the cap on the agarose bottle before placing it in the water bath. Water temperature should be at least 100 °C. Boil until the agarose is completely liquid. Remove the bottle from the waterbath and swirl occasionally.

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2. Melt Agarose (continued):

Microwave: Loosen the cap on the agarose bottle before microwaving. Heat in one-minute intervals on low to medium power until the agarose is melted.

NOTE: If, when heating in a microwave, the agarose starts to boil before melting completely, stop the microwave, swirl the agarose bottle, and continue to melt until the sample is completely liquid.

3. Cast Agarose Gels (Technique varies slightly depending on the type of equipment used.)

You may pour the gels in advance and store them refrigerated for up to one week prior to performing the electrophoresis portion of the lab.

a. Tape both ends of the gel-casting tray. (See Figure 1.) Be sure there is good adhesion between the surfaces of the tray and tape to avoid leaking. Strapping tape or similar is recommended. Alternatively, many gel box systems provide reusable attachments for both ends to avoid taping failures. Use as directed by the manufacturer.

b. Insert the well-forming comb into the slots at the end of the gel-casting tray. (See Figure 2.)

c. Pour approximately 15 mL of the molten agarose into the gel-casting tray. (See Figure 3.) Be sure that no liquid agarose leaks from the taped ends of the tray.

d. Allow the agarose to solidify. This will take approximately 20-30 minutes. Do not disturb the gel-casting tray or well-forming comb during this time.

e. After the agarose has solidified, remove the well-forming comb by gently pulling the comb up. Remove the tape from both ends of the gel-casting tray.

f. To store: Place the gel, on the casting tray, in a re-sealable bag and add 1-2 mL of 1X TBE buffer to the bag. Seal the bag and store it refrigerated until needed.

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notes

Pre-lAborAtory PrePArAtIon (ContInUed)

Figure 1

Figure 2

Figure 3(continued on next page)





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Pre-lAborAtory PrePArAtIon (ContInUed)

4. Prepare Dilute DNA Stain

NOTE: The DNA stain should be prepared just prior to staining the gel.

Add 5 mL Ward’s QUIKView DNA stain concentrate to 95 mL warm (50° - 55 °C) distilled water or tap water to obtain 100 mL of dilute stain.

5. Make copies of Student Guide.

Copy pages __ to __ of the student copymaster prior to starting class.

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Figure S1. Schematic gel. The open box at the top of columns A-C represents the loading wells where the DNA sample is loaded before the current is turned on. Column A represents DNA markers of known size after the current ran for 2 hrs. The distance that the bands migrate plotted on a semi log scale of the molecular weight in kilobases (kb) is a linear function so that the size of an unknown fragment can be estimated based upon the distance migrated. Column B represents a linearized plasmid with a shadow of uncut, supercoiled DNA just below it. The plasmid is estimated to be about 3.5 kb. Column C represents that same plasmid cut into two fragments with restriction endonucleases. Note that the sum of the fragment molecular weights adds up to the molecular weight of the linear plasmid.

objeCtIves

Justify the claim that humans

can manipulate heritable

information by identifying

at least two commonly used

technologies.

Pose questions about ethical,

social, or medical issues

surrounding human genetic

disorders [an application of

genetic engineering].

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bACkgroUndNucleic acid analysis has become a powerful tool in understanding organisms, their evolution, and their fundamental nature. Upon isolation of DNA from a cell source, that DNA may be examined using gel electrophoresis. Gel electrophoresis is a separation technology in which molecules are forced across a gel by an electrical current; activated electrodes at either end of the gel providing the driving force. The frictional force of the gel acts as a “molecular sieve”, separating the material by size. The substrate used primarily in the separation of large macromolecules, such as DNA or RNA, is agarose. Agarose is a natural colloid extracted from seaweed that, when melted and re-solidified, forms a matrix of microscopic pores. The size of these pores depends on the concentration of agarose used in the gel. Typically, the agarose concentration used in electrophoresis varies from 0.5% to 2.0%. The lower the concentration of agarose in a gel, the larger the pore size and the larger the nucleic acid fragments that can be separated. The length of a given DNA molecule can be determined by comparing the electrophoretic mobility on an agarose gel with that of a DNA marker sample of known length. (See Figure.S1.)





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bACkgroUnd (ContInUed)

notes


A DNA sample can be digested by restriction enzymes which cut double stranded DNA at specific sequences between 4 an 10 kb long. These restriction enzymes were purified from bacterial strains and are usually named based on an abbreviation of their purification source. For example, EcoR1 was purified from E.coli and specifically cuts at the sequence 5’-GAATTC-3’. EcoR1 is a restriction enzyme that leaves overhanging ends of sequence – it cuts between G and A on both strands. (See Figure S2.) Other endonucleases may cut to leave “blunt” ends that don’t have an overhang of single stranded nucleotides.

Figure S2: Restriction digestion example leaving overhanging sequence or “sticky ends” – EcoR1. If there was only one EcoR1 site in a circular plasmid, this cut would linearize the plasmid.

In the case of restriction digestion of very short (3 kb) and abundant bacterial plasmid DNA, it is easy to visualize individual bands and determine their size based upon their migration rates relative to those of known size fragments. (See Figure S1.)

A simple bacterial genome is about 1000 times larger than a plasmid while the human genome is about 3 x 109 base pairs or 106 times larger than a plasmid. Restriction enzyme digestion and agarose gel electrophoresis of genomic DNA similarly results in separation of the fragments according to size, however the individual bands are visible as a smear since there are so many bands of so many sizes.

Of the three billion nucleotides in human DNA, more than 99% are identical among individuals. The remaining 1% that is different,





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notes


however, adds up to a significant amount of code variations between individuals, making each person’s DNA profile as unique as a fingerprint. Due to the large number of possible variations, no two people (with the exception of identical twins) have the same DNA sequence.

For every 1,000 nucleotides inherited, there is one site of variation, or polymorphism. The DNA polymorphisms change the length of the DNA fragments produced by digestion with restriction enzymes. The exact number and size of fragments produced by a specific restriction enzyme digestion varies from person to person. The resulting fragments, called Restriction Fragment Length Polymorphisms (RFLPs), can be separated, and their size determined, by electrophoresis.

The difference in the fragments can be quantified to create a “DNA fingerprint”. Distinct RFLP patterns can be used to trace the inheritance of chromosomal regions with genetic disorders or to identify the origin of a blood sample in a criminal investigation. Scientists have identified more than 3,000 RFLPs in the human genetic code, many of which are highly variable among individuals. It is this large number of variable yet identifiable factors that allow scientists to identify individuals by the number and size of their various RFLPs.

Since human DNA has about 3 x 109 base pairs, digestion of the DNA with a restriction enzyme often produces tens or hundreds of thousands of DNA fragments. When run on an agarose gel, the sample will often appear as a long smear, with no resolution visible due to the extremely high number of fragments. One way to identify an individual RFLP band is by Southern Blot analysis. In this method, the DNA fragments in the agarose gel are transferred to a membrane, denatured into single strands and then hybridized with a smaller probe fragment of interest. The probe fragment will adhere to the membrane where it can bind to complementary sequences. The probe is labeled in some way (by radioactive isotope or luminescent molecule that provides a visual tag for the hybridization probe) then detected as a band of characteristic molecular weight on the membrane. Just as in restriction enzyme analysis, individual bands’ size can be determined through comparison to a known size standard, and by measuring migration distance relative to migration distance of known fragments.





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notes


The main characteristic of a probe fragment of interest for DNA fingerprinting would be one that identifies the genomic fragments that vary greatly between individuals. Most of the DNA in a chromosome is not used for the genetic code; it is uncertain what, if any, use this DNA may have. Because these regions are not essential to an organism’s development, it is more likely that changes will be found in these nonessential regions. The regions contain short nucleotide sequences that repeat from 20 to 100 times (e.g.,GTCAGTCAGTCAGTCA). The length of these short tandem repeat (STR) segments is highly variable in human populations so the size of restriction fragments carrying the repeats will be variable. These sequences are excellent hybridization probes since they identify specific RFLPs that are known to vary in size between individuals.

Often times only a small sample of DNA is available for analysis. Using the types of probes that are specific for targeted regions, those regions can be amplified for further analysis. The procedure is known as polymerase chain reaction (PCR). This concept was developed by Kary Mullis in 1983 and won him the Nobel Prize in 1993 because the technique is so powerful. It can be used to generate reliable DNA fingerprints from trace samples. In PCR, the variable fragments are not formed by restriction digestion at specific endonuclease sequences, but the fragments are built by amplifying the sequence between two specific hybridization sequences (which can be the same as the endonuclease sequences). A DNA fingerprint can be built by specifically amplifying regions that are highly variable between individuals.

The DNA fingerprinting technique is being used more and more frequently in legal matters. Using DNA fingerprinting, the identity of a person who has committed a violent crime can be determined from minute quantities of DNA left at the scene of the crime in the form of blood, semen, hair, or saliva. The DNA fingerprint matched to a suspect can be accurate to within one in 10 billion people, which is almost twice the total population in the world. Certain limitations in the technique prevent two samples from being identified as a “perfect match”, yet it is possible to measure the statistical probability of two samples coming from the same individual based on the number of known RFLPs that exist in a given population.





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notes DNA fingerprinting has many other applications, since half of a person’s genome comes from each parent, DNA fingerprinting can be used to determine familial relationships. It has a much higher certaint than a blood test when used to determine fatherhood in a paternity suit.

DNA fingerprinting can be used to track hereditary diseases passed down family lines and can be used to find the closest possible matches for organ transplants. It can be used to ascertain the level of inbreeding of endangered animals, aiding in the development of breeding programs to increase animals’ genetic health and diversity.





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sAfety PreCAUtIons CAUTION: The power supply used in this lab produces a high voltage that can cause severe electrical shock if handled improperly. For safe operation, follow all directions and precautions.

As general safe laboratory practice, it is recommended that you wear proper protective equipment, such as gloves, safety goggles, and a lab apron.

As general lab practice, read the lab through completely before starting, including any Material Safety Data Sheets (MSDSs) and live materials care sheets at the end of this booklet as well as any appropriate MSDSs for any additional substances you would like to test. One of the best sources is the vendor for the material. For example, when purchased at Ward’s, searching for the chemical on the Ward’s website will direct you to a link for the MSDS. (Note: There are no live materials care sheets included in this particular lab.)

At the end of the lab:

All laboratory bench tops should be wiped down with a 20% bleach solution or disinfectant to ensure cleanliness.

Wash your hands thoroughly with soap and water before leaving the laboratory.

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ProCedUre tIPs

When performing this lab activity, all data should be recorded in a lab notebook. You will need to construct your own data tables, where appropriate, in order to accurately capture the data from the investigation.

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PArt 1: ModelIng dnA And restrICtIon dIgests (strUCtUred InqUIry)

MAterIAls needed Per lAb groUP60 Red pop beads (phosphate)60 5-hole white beads (deoxyribose sugar)15 Orange pop beads (thymine)15 Yellow pop beads (adenine)15 Blue pop beads (cytosine)15 Green pop beads (guanine)6 Pink pop beads (radioactive phosphate)30 Plastic connectors (hydrogen bonds)1 Jan I restriction enzyme card1 Ward II restriction enzyme card1 Alkali card1 Probe card1 Suspect, victim, or murderer DNA strip (assigned by your instructor)1 Self-sealing bag

PArt 1 – sCenArIoHomicide investigators were called to the scene of a murder. They found bloodstains of two different blood types on the victim’s body. One of the stains was clearly the blood of the murderer.

Four suspects were apprehended, each of them with the same blood type. You and your fellow lab technicians (classmates) have been chosen to determine which of the four suspects most likely committed the crime. You will make use of several biotechnology procedures to accomplish your task. Be sure to follow the steps of the procedure precisely and pay attention to every detail. A mistake could result in the accusation of an innocent suspect or lead to the release of a violent criminal.

qqqqqqqqqqqqq

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ProCedUre – PArt 1: ModelIng dnA And restrICtIon dIgests notes


1. Assemble the simulated DNA molecule.

a. Construct a DNA molecule using as a blueprint the DNA strip you were assigned. Assemble the molecule as shown in the figure below. Be sure to assemble the beads in the exact sequence indicated on the strip to prevent incorrect results. Remember, thymine (orange) always pairs with adenine (yellow) and cytosine (blue) always pairs with guanine (green). The red and white pop beads form the sugar-phosphate backbone of the DNA molecule and the clear connectors represent the hydrogen bond formed between bases.

Figure S3

b. After your DNA molecule is assembled, double-check the sequence against the sequence on your DNA strip. Remember, accuracy is absolutely essential.

c. Place the DNA molecule on your desk or work area so that the 5’ TTT end is on the top left as shown below.

5’ 3’ TTT ...................................................................................... AAA ..................................................................................... 3’ 5’

NOTE: From this point on, be sure to always keep the DNA molecule in this orientation. Never allow the chain to be turned upside down or rotated. The 5’ TTT should always be on the top left of the molecule. If your chain should accidentally be moved, refer to the DNA strip to place it back in the proper orientation.

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2. Simulate restriction enzyme digest of the DNA molecule

a. Examine the restriction enzyme cards (Jan I and Ward II). Restriction enzymes are able to recognize and cut DNA at a specific DNA sequence. Note the dotted line on the restriction enzyme cards. These lines indicate the manner in which the DNA will be cut.

b. Begin with the enzyme labeled Jan I. Place the card on top of the left side of the DNA molecule, making sure the card is right side up. Move the card along the surface of the DNA molecule, from left to right, until the sequence on the card matches a sequence on the DNA molecule. When a match is found, stop and break the molecule apart in the manner indicated by the dotted line on the card.

NOTE: If the dotted line happens to lie on top of a bead, shift the molecule slightly until the dotted line lies between the beads.

c. Continue to move the restriction enzyme card along the molecule to the right, making sure to stop and break apart the molecule any time a sequence match is found. After you have completely digested the molecule with the enzyme, it is a good idea to double-check and make sure you have not missed any enzyme cut sites.

d. Repeat the procedure using the Ward II enzyme card. Be sure to keep the newly generated DNA fragments in their proper orientation when making the cuts in the molecule.

e. Examine your DNA molecule. The enzymes have broken your original DNA molecule into fragments of various sizes. The fragments are called Restriction Fragment Length Polymorphisms, or RFLPs for short.

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ProCedUre – PArt 1: ModelIng dnA And restrICtIon dIgests (ContInUed)notes





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3. Simulate gel electrophoresis of the DNA fragments.

You now have several DNA fragments of varying sizes. In order to examine them further, you must first separate the fragments based on their different sizes. To do this, you will now expose the DNA fragments to gel electrophoresis. Use the surface of your desk or work area as an agarose gel by reproducing the gel electrophoresis lanes in your Analysis sheet. Evenly divide the entire length of your desk or work area into numbered sections, with the largest number (22) at the top and the smallest number (2) at the bottom. Small pieces of numbered tape or a long strip of paper may be the easiest way to do this.

The top of your gel lane (22) will represent the negative (-) end of the electrophoresis chamber and the bottom end (2) will represent the positive (+) end. Since DNA in an aqueous solution has a sugar-phosphate backbone that is highly negatively charged, that DNA exposed to an electrical current will always migrate toward a positive charge. Using a small piece of tape, mark the top of the lane (-) and the bottom of the lane (+).

The gel used in electrophoresis contains millions of microscopic pores. When exposed to electricity, the DNA fragments move through these pores. Smaller DNA fragments are able to wind their way through the pores more easily, moving farther in a set amount of time than DNA fragments of a larger size. Starting with your smallest DNA fragment, place it at the top of your gel lane. Slide the fragment along the gel lane until the number of bases in the top strand of the DNA molecule matches the number labeled on your gel lane. For example, if the smallest DNA fragment has four bases on the top strand, slide the fragment down the gel lane until it reaches the spot marked four and leave it in that position.

Continue to separate your DNA fragments in this manner. Work from smallest to largest fragment until they are all properly positioned along the length of your gel lane.






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Steps 4 and 5 are optional. These steps model Southern Blot Techniques used in fingerprint analysis. If you choose not to do Steps 4 and 5, go directly to Step 6 (on page S ___.

4. Optional Simulated Denaturing and Probing the DNA Fragments (continued)

a. Since restriction enzyme digestion of actual human DNA may yield tens or hundreds of thousands of fragments, a probe is usually applied to RFLPs to select only certain DNA fragments for analysis. The probe is a small piece of DNA with a radioactive marker that will show up when exposed to X-ray film. In order to hybridize (attach) the probe to DNA fragments, they must first be separated into single stranded molecules. This is referred to as denaturing the DNA. Denature your DNA fragments by passing the alkali card over each fragment. The alkali card represents a chemical (such as sodium hydroxide) that will separate the DNA strands. Remove the bottom strand of each DNA fragment (including the hydrogen bonds) and set it aside.

NOTE: The alkali cards are not absolutely necessary in the completion of the exercise. They are simply included to reinforce the concept that the DNA is exposed to a treatment in order to be denatured.

b. Construct two radioactive probes using six pink beads (radioactive phosphate), six white beads (deoxyribose), two orange beads (thymine), two green beads (guanine), and two blue beads (cytosine). Use the figure below as a guide when assembling your probes.

Figure S4

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4. Optional Simulated Denaturing and Probing the DNA Fragments (continued)

c. Using a probe card, probe the single-stranded DNA fragments in your gel. Search the DNA fragments for a sequence complementary to the sequence found in the probes. When you encounter a sequence complementary (5’ A C G 3’) to the sequence of the probe (3’ T G C 5’), use three hydrogen bonds to attach the probe to the DNA fragment containing the complementary sequence. Be sure to probe all of the DNA fragments in your gel.

NOTE: In an actual Southern blotting procedure, the DNA is transferred from the gel to a membrane, usually nylon or nitrocellulose, and then the membrane is exposed to the probes. The membrane is more durable and easier to handle than the flimsy gel. This is not necessary in this simulation so this step is omitted.

5. Optional Simulated Autoradiography

a. In order to examine the DNA fragments to which a probe has attached, a piece of X-ray film is laid on top of the membrane. The radioactive phosphate in the probe will cause the X-ray film to develop in that region, resulting in a dark band on the film. To simulate this, examine the size of the DNA fragments to which a probe has attached. Sketch dark bands for your suspect (or murderer or victim) in the gel lanes in the Analysis section of the lab. If, for example, you have a DNA fragment ten bases long with a probe attached, sketch a dark band across the gel lane at the ten marking. If you have a fragment that is seventeen bases long, sketch a band halfway between the eighteen and sixteen on the gel lane.

b. After you have completed the autoradiograph, share your results with the rest of the teams in the class. Also, copy the results from the other suspects, murderer, and victim using the results obtained by the other groups.

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6. Sketch the model you created. Using this model, match the DNA fingerprint pattern of the suspects to the DNA fingerprint from the scene of the crime. Who is guilty?

Suspect4DNAfingerprintmatchesthemurderscenesample.


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PArt 2 – gUIded InqUIry: AgArose gel eleCtroPhoresIs

MAterIAls needed Per lAb groUP1 0.8% agarose gel, on gel tray200 mL 1X TBE running buffer100 mL DNA stain1 Staining trayMicropipets1 Metric ruler1 Calculator

Shared Materials:

DNA SamplesTube #1: DNA Marker StandardTube #2: Crime Scene DNA SampleTube #3: Suspect 1 DNA SampleTube #4: Suspect 2 DNA Sample

PArt 2 – sCenArIoInvestigators were called to the scene of a burglary where it appeared that as the burglar rushed to leave, he ran into a glass door, cutting his arm and tearing his shirt. The investigators removed small pieces of bloodstained fabric from the door to be tested; the blood sample was determined to be type A. Two suspects were apprehended; unfortunately, both had type A blood. Investigators have now resorted to DNA fingerprinting to determine which of the two suspects is the burglar.

qqqqqqq

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ProCedUre tIPs

When performing this lab activity, all data should be recorded in a lab notebook. You will need to construct your own data tables, where appropriate, in order to accurately capture the data from the investigation.

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CAUtIon !

The power supply used in this lab produces a high voltage that can cause severe electrical shock if handled improperly. For safe operation, follow all directions

and precautions.

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notes

PArt 2 – ProCedUre: AgArose gel eleCtroPhoresIs

1. Load a gel.

Use one of the following two methods to load each DNA sample into the corresponding gel well listed below.

Lane #1: DNA Marker Standard

Lane #2: Crime Scene DNA Sample

Lane #3: Suspect 1 DNA Sample

Lane #4: Suspect 2 DNA Sample

NOTE: The amount of DNA in the reaction tubes is extremely small.

Method 1: Dry

a. Place the tray with the gel on the lab bench.

b. Use a micropipet to load 10 µL of each DNA sample into the corresponding lane. Do not pierce the bottom of the wells with the micropipet tip. Do not overload wells.

c. Place the tray with the loaded gel in the center of the electrophoresis chamber with the wells situated closest to the negative (black) electrode (Figure S5).

Figure S5

NOTE: When filling the electrophoresis chamber with buffer, avoid pouring the solution directly onto the gel and be sure to pour it VERY SLOWLY. If the buffer is poured too quickly, it may wash away the DNA samples.

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Method 1: Dry (continued)

d. Add approximately 200 mL of 1X TBE running buffer to the chamber: Slowly pour buffer from a beaker into one side of the chamber until the buffer is level with the top of the gel. Continue to slowly add buffer until the level is approximately 2-3 mm above the top of the gel.

NOTE: The current in the chamber is carried by platinum wires that extend along the bottom of the chamber. Be sure to fill your chamber until the buffer level is a couple of millimeters over the top of the gel. The buffer should not be touching the electrode junctions at the top of the chamber.

NOTE: If the gel and casting tray begin to float, use a pipet to clear the air hole that is located on top of the casting tray.

Method 2: Submarine

a. Place the gel, on the gel tray, in the center of the electrophoresis chamber with the wells closest to the negative (black) electrode.

b. Add approximately 200 mL of 1X TBE running buffer to the chamber. Slowly pour buffer from a beaker into one side of the chamber until the buffer is level with the top of the gel. Continue to slowly add buffer until the level is approximately 2-3 mm above the top of the gel.

Note: The current in the chamber is carried by platinum wires that extend along the bottom of the chamber. Be sure to fill your chamber until the buffer level is a couple of millimeters over the top of the gel. The buffer should not be touching the electrode junctions at the top of the chamber.

Note: If the gel and casting tray begin to float, use a pipet to clear the air hole that is located on top of the casting tray.

c. Use a micropipet to load 10 µL of each DNA sample into the corresponding lane. Do not pierce the bottom of the wells with the micropipet tip. Do not overload wells.

Note: Do not overfill the chamber. Wipe off any spills.

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2. Run the gel.

a. Making sure the cover is dry, place it onto the electrophoresis chamber. Wipe off any spills on the apparatus before proceeding to the next step.

b. Connect the power supply. Making sure that the patch cords, as well as the female jacks on the chamber, are completely dry, connect the red patch cord to the red terminal on the power supply. Connect the black patch cord to the black terminal on the power supply.

NOTE: Check the connections before proceeding to the next step.

c. Once the power supply is connected to the patch cords, bubbles will form along the platinum electrodes.

d. Observe the migration of the tracking dye down the gel toward the red (positive) electrode. Disconnect the power supply when the tracking dye has reached the end of the gel.

3. Visualize the bands.

a. Lift the gel tray with the gel from the chamber, and gently push the gel off the gel tray into a staining tray.

Optional stopping point: If the lab period does not allow time to stain and destain the gel, measure the distance the loading dye has traveled (measure to the center of the diffuse band formed by the loading dye) on the gel, and record this measurement in Tables 1-4. Be sure to take this measurement, as the loading dye will diffuse out of the gel within a couple of hours. Place the gel in a resealable bag, and add 1-2 mL of 1X TBE buffer. Store the gel refrigerated until the next lab period.

b. Wearing protective gloves, pour approximately 100 mL of warm dilute stain into the staining tray so the stain just covers the gel.

c. Let the gel stain for approximately 30-45 minutes.

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ProCedUre – PArt 2: AgArose gel eleCtroPhoresIs (ContInUed)notes


3. Visualize the bands (continued)

d. Carefully decant the used stain. Make sure the gel remains flat and does not move up against the corner. Decant the stain directly to a sink drain and flush with water.

NOTE: The dilute DNA stain may be saved and reused several times. For best results, reheat the stain before using.

e. Add distilled or tap water to the staining tray. To accelerate destaining, gently rock the tray. Destain until bands are distinct, with little background color. This will take between 20 and 30 minutes, depending on the amount of agitation. Change the water several times, or destain the gel, without changing the water, overnight.

f. View the gel against a light background, such as white paper, or on a light table. In the Analysis section, roughly sketch the bands you see on the blank gel.

g. Gels can be stored in self-sealing plastic bags. For long-term storage, add several drops of dilute stain to the bag to prevent the DNA bands from fading . If fading does occur, the gel can be restained using the above procedure.

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ProCedUre – PArt 2: AgArose gel eleCtroPhoresIs (ContInUed)


4. Analyze your gel.

a. Measure the distance of the DNA bands, in millimeters, from the bottom of the sample well to the bottom of each DNA fragment on the marker. Measuring to the bottom of each fragment band ensures consistency and accurate measurements. Do not measure the migration distance of the largest fragment nearest the well; it will not be on the standard curve and will skew results.

Record the measurements in Table 1 (on the next page)

b. On semi-log graph paper, plot a standard curve for the DNA marker standard. Plot the migration distance in millimeters on the X-axis, against the molecular size in base pairs (bp) of each fragment. Draw the best-fit line to your points.

NOTE: When plotting on semi-log graph paper, the fragment size is expressed as a logarithmic scale. Label the first series of lines 100 bp, 200 bp, 300 bp, etc. Then label the second series of lines 1,000 bp, 2,000 bp, 3,000 bp, etc. The third series would be 10,000 bp, 20,000 bp, etc.

c. Measure the distance that each band traveled for the lanes containing the two suspects and the crime scene DNA. Record the data in the appropriate sections of Table 1.

d. Calculate the base pair size of each of the fragments by moving along the X-axis until you have reached the distance traveled by the fragment. From that point, move upward until you intersect the line of best fit on the graph. Determine where that point is on the Y-axis and estimate the base pair value at that point. Enter the values in the table, in units of kilobases (kb).

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dAtA – PArt 2: AgArose gel eleCtroPhoresIs

Band # Distance migrated from well (mm) Molecular weight (kb)

Standard Top No measurement

Standard band 1 (first) 9.4

ST 2 6.6

ST 3 4.4

ST 4 2.3

ST 5 2.0

Crime scene Band 1 (top)

CS 2

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Suspect 1 band 1 (top)

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S1 10

Suspect 2 band 1

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Table 1





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Answer key – PArt 1: ModelIng dnA And restrICtIon dIgests





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Answer key – PArt 2: AgArose gel eleCtroPhoresIs The results shown below are based on sample data. Your students may have similar results.

Band # Distance migrated

from well (mm)

Molecular weight

(Kilobase or kb)

Standard Top No measurement No measurement

Standard band 1 (first) 28 9.4 kb

ST 2 32 6.6

ST 3 38 4.4

ST 4 49 2.3

ST 5 52 2.0

Crime scene band 1 (top) No measurement No measurement

CS 238

5.1, 5.0

(appear as one band)

CS 3 40 4.3

CS 4 43 3.5

CS 5 55 2.0

CS 6 58 1.9

CS 7 61 1.6

CS 8 65 1.4

CS 9 74 0.9

CS 10 76 0.8

Suspect 1 band 1 (top) No measurement No measurement

S1 2 33 7.4

S1 335

5.8, 5.6


S1 4 38 4.9

S1 5 43 3.5

S1 6 No measurement No measurement





Suspect 2 band 1 No measurement No measurement

S2 238

5.1, 5.0


S2 3 40 4.3

S2 4 43 3.5

S2 5 55 2.0

S2 6 58 1.9

S2 7 61 1.6

S2 8 65 1.4

S2 9 74 0.9

S2 10 76 0.8





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AssessMent qUestIons for PArts 1 And 21. Based on the class results, which of the suspects in Part 1 is the murderer? Explain your answer.

Suspect #4. The students should note that the autoradiograph for suspect #4 is identical to that of the murderer’s autoradiograph.

2. Compare the banding patterns formed on each lane of the gel in Part 2. Do you think the three DNA samples tested are the same? Explain. How can you further verify whether or not any of the DNA samples tested are the same?

Two of the three DNA samples tested are identical; they have the same banding pattern. To further verifywhetherornotanyoftheDNAsamplestestedarethesame,theDNAfingerprintingprotocolcould be followed by a procedure called sequencing. DNA sequencing would allow the determination of the exact DNA sequence of the DNA fragments on the gel.

3. Which of the two suspects in Part 2 do you believe is the real burglar? Explain your answer.

Suspect 2’s DNA banding pattern closely matches the DNA pattern of the crime scene DNA sample. It can be concluded that, based on the similarities of these two samples, suspect 2 committed the crime.

4. Each of the following plays an important role in the process of DNA fingerprinting. Explain the function of each.

Restriction enzymes –

RestrictionenzymescutDNAmoleculesonlyatspecificsiteswithinthemolecule,resultinginDNAfragments of varying size. Since the sequence of DNA differs from individual to individual, the same restriction enzyme applied to the DNA from two different individuals will result in differences in both the number of DNA fragments and the sizes of the DNA fragments in the two DNA samples.

Gel electrophoresis –

Gel electrophoresis is a technology that allows the separation of DNA fragments, generated by restriction enzymes, based on size. By separating the fragments from largest to smallest, scientists are able to more easily discriminate the differences between two different DNA samples.

DNA probes –

ADNAprobeallowstheidentificationofspecificDNAfragmentsinasample.SinceaDNAsample,digested with restriction enzymes, may often yield hundreds of thousands of DNA fragments, a tool is necessarytofurtherisolateandidentifyspecificfragmentswiththedigestedDNAsample.

Autoradiograph –

ThispieceofX-rayfilmshowsexactlywhereinthesampletheprobeshaveattachedtoDNAfragments.Once a probe is attached to a DNA fragment, a scientist still has no idea where in the sample the probe hasattacheduntiltheX-rayfilmisdevelopedbytheradioactivemarkerswithinthesample.






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5. Suppose you had an enzyme that recognized a sequence of six nucleotides. What are the odds that this sequence would appear in a random chain of DNA?

1 in 4,096 (4 x 4 x 4 x 4 x 4 x 4). Since there are four bases (A, C, G, and T) that could occupy a position in the molecule and they must appear in a six base sequence.

6. Given the answer to the above question, suppose you have a piece of human DNA that is three billion base pairs in length. How many fragments will be generated by digesting the DNA with the above enzyme?

3,000,000,000 divided by 4,096, or approximately 732,400 DNA fragments.

7. EcoR I recognizes GAATTC, and Hind III recognizes AAGCTT. A student adds EcoR I to a genomic DNA sample. To another quantity of the same DNA, she adds Hind III. In a third tube she adds both enzymes. She runs a gel and the following occurs:

Hind III EcoR I Double

———

———– ———–

———–

———–

——— ———–

After viewing the gel, the student draws the following map of the genomic DNA

a. Explain why she placed the nucleotides as she did.

The EcoR I cut in one place which is why there are two bands. One fragment is slightly smaller than the other.

AssessMent qUestIons for PArts 1 And 2 (ContInUed)






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7. (continued)

After a little more thought, she added another sequence to the DNA:

b. Explain how she was able to add this sequence in this position based on the results of her gel.

The Hind III by itself only cut once. The EcoR I also cut once. In the double digest, the Hind III cut the smaller of the EcoR I fragments.

8. You have used electrophoresis to perform DNA fingerprinting and identify a guilty suspect involved in a criminal investigation. Research another use for electrophoresis and briefly describe the benefits of using this technology.

Answers will vary. Students may present topics such as paternity testing, screening for genetic disorders,proteinidentification,studyingevolutionaryrelationships,geneticengineering,etc.

9. The application of DNA fingerprinting technology in forensic science as well as in medicine has raised many legal and ethical concerns. Discuss the following questions: Should data banks be established for DNA information? What are some benefits to society and individuals of DNA data banks? What are some of the risks to society and individuals of establishing a DNA databank?

Answers will vary.

10. In your lab, you ran your DNA samples on a 0.8% agarose gel. Would you get the same results if you ran your samples on a higher percentage agarose gel? Why or why not?

Student answers will vary. Running the samples on a gel of only slightly higher percentage of agarose (0.9%or1.0%)wouldprobablynotaffecttheresultssignificantly.However,ifthepercentageweregreatly increased, such as 2.0% or 2.5%, the larger fragments may not be able to move through the decreased pore size, resulting in less effective electrophoretic separation.

11. Predict what would happen if you place your gel in the electrophoresis chamber with the wells containing the DNA next to the red electrode instead of the black?

Students should realize that the DNA would still be attracted to the positive electrode. The DNA samples would run quickly toward the top of the gel and out into the buffer.







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12. Below is a list of the components involved in agarose gel electrophoresis. Briefly describe the purpose of each component.

Agarose gel – the substrate used to separate molecules. Contains microscopic pores that allow smaller molecules to move through faster.

TBE buffer – a liquid buffer that protects the DNA molecules and allows electricity to move through the chamber, driving the molecules across the gel.

Electrophoresis chamber – holds all of the components of the separation. Creates a closed system in which to submerge a gel and apply electrical current.

Power supply – the unit generating the electricity being applied to the chamber.

DNA samples – the substance to be separated. A mixture of differently-sized fragments that separate when electricity is applied.

DNA stain – used to stain the gel after electrophoresis. Since DNA cannot be seen with the naked eye, DNA stain is necessary to visualize your DNA fragments.






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PArt 3– genetICs And InforMAtIon trAnsfer: restrICtIon enzyMe

oPen InqUIry: desIgn An exPerIMentWhat questions occurred to you as you performed the DNA fingerprinting protocols? Now that you are familiar with DNA fingerprinting and agarose gel electrophoresis, design an experiment to investigate one of your questions. You may consider the following in your investigation:

How do the results of DNA fingerprinting change as different restriction enzymes are used?

How do changes in certain parameters of restriction digests or electrophoresis affect results of DNA fingerprinting?

Before starting your experiment, plan your investigation in your lab notebook. Have your teacher check over and initial your experiment design. Once your design is approved, investigate your hypothesis. Be sure to record all observations and data in your laboratory sheet or notebook.

Use the following steps when designing your experiment.

1. Define the question or testable hypothesis.

2. Describe the background information. Include previous experiments.

3. Describe the experimental design with controls, variables, and observations.

4. Describe the possible results and how they would be interpreted.

5. List the materials and methods to be used.

6. Note potential safety issues.

After the plan is approved by your teacher:

7. The step by step procedure should be documented in the lab notebook. This includes recording the calculations of concentrations, etc. as well as the weights and volumes used.

8. The results should be recorded (including drawings, photos, data print outs).

9. The analysis of results should be recorded.

exPerIMent desIgn tIPs

The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following ques-tions, which also might arise in peer review:

Explain the purpose of a procedural step.

Identify the independent variables and the dependent variables in an experiment.

What results would you expect to see in the control group? The experimental group?

How does XXXX concept account for YYYY findings?

Describe a method to determine XXXX.

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10. Draw conclusions based on how the results compared to the predictions.

11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance and uncontrolled variables.

12. Further study direction should be considered.

notes

PArt 3: oPen InqUIry (ContInUed)





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restrICtIon enzyMe CArds

Kit # 36-7409

Restriction Enzyme Card

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Probe Card

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Suspect/Victim DNA Sample Strips

Cut along dotted lines

5’ 3’T T T C A G G G A T G T A T G A C G T G G A C G T A T C G G

A A A G T C C C T A C A T A C T G C A C C T G C A T A G C C 3’ 5’

Murderer

5’ 3’T T T A T A C G T C G T G G A C G A T G G T G C T G A G A G

A A A T A T G C A G C A C C T G C T A C C A C G A C T C T C 3’ 5’

Suspect 1

5’ 3’T T T A T A C G T C G T G G A C G A T C G T G C T G A G A G

A A A T A T G C A G C A C C T G C T A G C A C G A C T C T C 3’ 5’

Suspect 2

5’ 3’T T T C T G G G A T G T A T G A C G C T A G T G G A C G A G

A A A G A C C C T A C A T A C T G C G A T C A C C T G C T C 3’ 5’

Suspect 3



Suspect 4

5’ 3’T T T A T A C G T C G T G G A T C A T G G T C G A C G G A G

A A A T A T G C A G C A C C T A G T A C C A G C T G C C T C 3’ 5’

Victim





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Murderer



Suspect 1



Suspect 2



Suspect 3



Suspect 4



Victim





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Murderer



Suspect 1



Suspect 2



Suspect 3



Suspect 4



Victim





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250-7458 v.5/12

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MAterIAl sAfety dAtA sheets

Material Safety Data SheetMay be used to comply with OSHA's Hazard CommunicationStandard. 29 CFR 1910.1200 Standard must be consulted for

specific requirements.

IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.

Section IManufacturer's Name

Section II - Hazardous Ingredients/Identify Information

Emergency Telephone Number

Telephone Number for information

Date Prepared

Signature of Preparer (optional)

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

EDVOTEK, Inc.

14676 Rothgeb DriveRockville, MD 20850

Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV

Other Limits Recommended % (Optional)

(301) 251-5990

(301) 251-5990

Boiling Point

Section III - Physical/Chemical Characteristics

Unusual Fire and Explosion Hazards

Special Fire Fighting Procedures

Vapor Pressure (mm Hg.)

Vapor Density (AIR = 1)

Solubility in Water

Appearance and Odor

Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)

Specific Gravity (H 0 = 1) 2

EDVOTEK®

FlashBlue™

03/30/10

Methylene Blue

3.7 Bis (Dimethylamino) Phenothiazin 5 IUM Chloride No data availableCAS # 61-73-4

No data

No data

No data

No data

No data

No data

Soluble - cold

Dark purple color, no odor

No data available No data No data

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

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

Emits toxid fumes under fire conditions

Stability

Section V - Reactivity DataUnstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion?Skin?

Other

Stable

HazardousPolymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

X None

Strong oxidizing agents

Toxic fumes of Carbon monoxide, Carbon dioxide, nitrogen oxides, sulfur oxides, hydrogen, chloride gas

X None

Yes Yes Yes

Skin: May cause skin irritation Eyes: May cause eye irritation Inhalation: Cyanosis

Meets criteria for proposed OSHA medical records rule PEREAC 47.30420.82

No data available

No data available

Treat symptomatically

Ventilate area and wash spill site

Mix material with a combustible solvent and burn in chemical

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

Keep tightly closed. Store in cool, dry place

None

MIOSH/OSHA approved, SCBA

Required

Rubber Chem. safety goggles

Rubber boots





250-7458 v.5/12

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Section 2 Composition / Information on Ingredients

Product

Chemical Name

MATERIAL SAFETY DATA SHEET

CHEMTREC 24 Hour Emergency Phone Number (800) 424-9300

CAS # % TLV Units

Emergency Overview

The information contained herein is furnished without warranty of any kind. Employers should use this information only as a supplementto other information gathered by them and must make independent determinations of suitability and completeness of information from allsources to assure proper use of these materials and the safety and health of employees. * Hazardous Materials Industrial Standards.

Section 1 Chemical Product and Company Information

Section 2 Composition / Information on Ingredients

Section 3 Hazards Identification

Section 5 Fire Fighting Measures

Section 6 Accidental Release Measures

Section 7 Handling & Storage

Section 8 Exposure Controls / Personal Protection

Section 12 Ecological Information

Section 13 Disposal Considerations

Section 14 Transport Information

Section 16 Additional Information

NFPA0 = Minimal1 = Slight2 = Moderate3 = Serious4 = Severe

Synonyms

Section 15 Regulatory Information

HMIS *

0 = Minimal1 = Slight2 = Moderate3 = Serious4 = Severe

HealthFireReactivityContact

Section 9 Physical & Chemical Properties

Section 10 Stability & Reactivity

Section 11 Toxicological InformationSection 4 First Aid Measures

LL0248March 1, 2010James A. Bertsch

GENERAL STORAGE CODE GREEN

LOADING DYE 10X

Bromophenol blue 62625-28-9 0.12% None established.Sucrose 57-50-1 50.0% None established.Tris tromethamine 77-86-1 0.12% None established.Water 7732-18-5 49.63% None established.

(ACGIH 2001)

CAUTION!MAY BE HARMFUL IF SWALLOWED. MAY CAUSE IRRITATION.Keep container in a cool, well-ventilated place. Protect from light. Washthoroughly after handling. Target organs: None known.

General information: In fire conditions, wear a NIOSH/MSHA-approved self-contained breathing apparatus andfull protective gear. During a fire, irritating and highly toxic gases may be generated by thermal decomposition orcombustion.

Use proper personal protective equipment as indicated in Section 8. Absorb with inert dry material, sweep orvacuum up and place in a suitable container for proper disposal. Wash spill area with soap and water. Avoid runoffinto storm sewers and ditches which lead to waterways.

Effects of overexposure: May be harmful by ingestion or skin absorption. May cause irritation. To the best ofour knowledge, the chemical, physical and toxicological properties have not been thoroughly investigated.Specific data is not available. Exercise appropriate procedures to minimize potential hazards.

ORL-RAT LD50: N/AIHL-RAT LC50: N/ASKN-RBT LD50: N/A

Data not yet available.

These disposal guidelines are intended for the disposal of catalog-size quantities only. Federal regulations mayapply to empty container. State and/or local regulations may be different. Dispose of in accordance with all local,state and federal regulations or contract with a licensed chemical disposal agency.

UN/NA number: N/AShipping name: Not Regulated.Hazard class: N/APacking group: N/AExceptions: N/A

INGESTION: Call physician or Poison Control Center immediately. Induce vomiting only if advised by appropriatemedical personnel. Never give anything by mouth to an unconscious person.

INHALATION: Remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen.Get medical attention.

EYE CONTACT: Check for and remove contact lenses. Flush thoroughly with water for at least 15 minutes, liftingupper and lower eyelids occasionally. Get immediate medical attention.

SKIN CONTACT: Remove contaminated clothing. Flush thoroughly with mild soap and water. If irritation occurs,get medical attention.

Extinguishing Media: Carbon dioxide, dry chemical, water spray, alcohol foam.Flash Point: Non-flammable.Autoignition temperature: N/AExplosion Limits: Lower: N/A Upper: N/A

MSDS No.:Revision Date:Approved by:

N/A

All components listed with the following agencies: TSCA, EINECS, DSL.

1001

Physical state: Liquid.Appearance: Clear blue.Odor: No odor.pH: N/AVapor pressure (mm Hg): 14 (water)Vapor Density (Air = 1): 0.7 (water)Evaporation rate (Water = 1): < 1Viscosity: N/A

Chemical stability: Stable Hazardous polymerization: Will not occur.Conditions to avoid: Excessive temperatures and heat.

Incompatibilities with other materials: Strong oxidizers.Hazardous decomposition products: Carbon oxides, sulfur oxides and bromides.

Boiling point: ~100°C (212°F) (water)Freezing / Melting point: ~0°C (~32°F) (water)Decomposition temperature: N/ASolubility: Complete.Specific gravity (H2O = 1): ~ 1.0Percent volatile (%): 49.63%Molecular formula: Mixture.Molecular weight: Mixture.

MSDS No.: LL0248

None listed.

Engineering controls: Facilities storing or utilizing this material should be equipped with an eyewash facility anda safety shower and fire extinguishing material. Personnel should wear safety glasses, goggles, or faceshield,lab coat or apron, appropriate protective gloves. Use adequate ventilation to keep airborne concentrations low.

Respiratory protection: None should be needed in normal laboratory handling at room temperatures. If mistyconditions prevail, work in fume hood or wear a NIOSH/MSHA-approved respirator.

Read label on container before using. Do not wear contact lenses when working with chemicals. Keep containertightly closed. For laboratory use only. Not for drug, food or household use. Keep out of reach of children.Handling: Use with adequate ventilation. Avoid contact with eyes, skin and clothing. Avoid ingestion. Do notinhale vapors, spray or mist. Wash thoroughly after handling. Remove and wash clothing before reuse.Storage: Store in a cool, dry, well-ventilated area away from incompatible substances. Protect from light.





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MAterIAl sAfety dAtA sheetsMaterial Safety Data Sheet Page 1 of 2

© 2008, Scholar Chemistry. All Rights Reserved. 12/20/2011

Agarose MSDS # 15.10 Section 1: Product and Company Identification

Agarose Synonyms/General Names: N/A Product Use: For educational use only Manufacturer: Columbus Chemical Industries, Inc., Columbus, WI 53925.

24 Hour Emergency Information Telephone Numbers CHEMTREC (USA): 800-424-9300 CANUTEC (Canada): 613-424-6666

ScholAR Chemistry; 5100 W. Henrietta Rd, Rochester, NY 14586; (866) 260-0501; www.Scholarchemistry.com

Section 2: Hazards Identification White to tan powder; bland characteristic odor. HMIS (0 to 4) This material is not considered hazardous Target organs: Liver. This material is not considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200) if used properly.

Section 3: Composition / Information on Ingredients Agarose (9012-36-6), 100% Section 4: First Aid Measures

Always seek professional medical attention after first aid measures are provided. Eyes: Immediately flush eyes with excess water for 15 minutes, lifting lower and upper eyelids occasionally. Skin: Immediately flush skin with excess water for 15 minutes while removing contaminated clothing. Ingestion: Call Poison Control immediately. Rinse mouth with cold water. Give victim 1-2 cups of water or milk to drink. Induce vomiting immediately. Inhalation: Remove to fresh air. If not breathing, give artificial respiration.

Section 5: Fire Fighting Measures When heated to decomposition, emits acrid fumes. 0 Protective equipment and precautions for firefighters: Use foam or dry chemical to extinguish fire. 0 0

Firefighters should wear full fire fighting turn-out gear and respiratory protection (SCBA). Cool container with water spray. Material is not sensitive to mechanical impact or static discharge.

Section 6: Accidental Release Measures Use personal protection recommended in Section 8. Isolate the hazard area and deny entry to unnecessary and unprotected personnel. Sweep up spill and place in sealed bag or container for disposal. Wash spill area after pickup is complete. See Section 13 for disposal information.

Section 7: Handling and Storage Green Handling: Use with adequate ventilation and do not breathe dust or vapor. Avoid contact with skin, eyes, or clothing. Wash

hands thoroughly after handling. Storage: Store in General Storage Area [Green Storage] with other items with no specific storage hazards. Store in a cool, dry,

well-ventilated, locked store room away from incompatible materials.

Section 8: Exposure Controls / Personal Protection Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Use NIOSH-approved respirator with a dust cartridge. Exposure guidelines: Agarose: OSHA PEL: N/A, ACGIH: TLV: N/A, STEL: N/A.

Health 0 Fire Hazard 1 Reactivity 0






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MSDS # 15.10 Agarose Scholar Chemistry Section 9: Physical and Chemical Properties Molecular formula N/A. Appearance White to tan powder. Molecular weight N/A. Odor Bland characteristic odor. Specific Gravity N/A. Odor Threshold N/A. Vapor Density (air=1) N/A. Solubility Soluble in boiling water . Melting Point 260°C. Evaporation rate N/A (Butyl acetate = 1). Boiling Point/Range N/A. Partition Coefficient N/A (log POW). Vapor Pressure (20°C) N/A. pH N/A. Flash Point: N/A. LEL N/A. Autoignition Temp.: N/A. UEL N/A.

N/A = Not available or applicable

Section 10: Stability and Reactivity Avoid heat and moisture. Stability: Stable under normal conditions of use and storage. Incompatibility: Strong oxidizers Shelf life: Indefinite if stored properly.

Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, conjunctivitis. Skin: Redness, itching. Ingestion: Irritation and burning sensations of mouth and throat, nausea, vomiting and abdominal pain. Inhalation: Irritation of mucous membranes, coughing, wheezing, shortness of breath, Chronic Effects: No information found. Sensitization: none expected Agarose: LD50 [oral, rat]; 2260 mg/kg; LC50 [rat]; N/A; LD50 Dermal [rabbit]; N/A Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.

Section 12: Ecological Information Ecotoxicity (aquatic and terrestrial): Ecological impact has not been determined.

Section 13: Disposal Considerations Check with all applicable local, regional, and national laws and regulations. Local regulations may be more stringent than regional or national regulations. Small amounts of this material may be suitable for sanitary sewer or trash disposal.

Section 14: Transport Information DOT Shipping Name: Not regulated by DOT. Canada TDG: Not regulated by TDG. DOT Hazard Class: Hazard Class: Identification Number: UN Number:

Section 15: Regulatory Information EINECS: Listed (232-731-8) . WHMIS Canada: Not WHMIS Controlled. TSCA: All components are listed or are exempt. California Proposition 65: Not listed. The product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS contains all the information required by the Controlled Products Regulations.

Section 16: Other Information Current Issue Date: December 20, 2011 Disclaimer: Scholar Chemistry and Columbus Chemical Industries, Inc., (“S&C”) believes that the information herein is factual but is not intended to be all inclusive. The information relates only to the specific material designated and does not relate to its use in combination with other materials or its use as to any particular process. Because safety standards and regulations are subject to change and because S&C has no continuing control over the material, those handling, storing or using the material should satisfy themselves that they have current information regarding the particular way the material is handled, stored or used and that the same is done in accordance with federal, state and local law. S&C makes no warranty, expressed or implied, including (without limitation) warranties with respect to the completeness or continuing accuracy of the information contained herein or with respect to fitness for any particular use.





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Material Safety Data Sheet Page 1 of 2


Tris-Borate-EDTA MSDS # 778.00 Section 1: Product and Company Identification

Tris-Borate-EDTA Synonyms/General Names: TBE 5X Buffer Product Use: For educational use only Manufacturer: Columbus Chemical Industries, Inc., Columbus, WI 53925.

24 Hour Emergency Information Telephone Numbers CHEMTREC (USA): 800-424-9300 CANUTEC (Canada): 613-424-6666

ScholAR Chemistry; 5100 W. Henrietta Rd, Rochester, NY 14586; (866) 260-0501; www.Scholarchemistry.com

Section 2: Hazards Identification Clear colorless liquid, no odor. HMIS (0 to 4) CAUTION! Slightly toxic by ingestion and body tissue irritant. Target organs: None known. This material is considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200).

Section 3: Composition / Information on Ingredients Tris-Hydroxymethylaminomethane (77-86-1), 5-6%. Boric Acid (10043-35-3), 2-4%. EDTA, Disodium Salt (6381-92-6), < 1%. Water (7732-18-5), 90-94%. Section 4: First Aid Measures

Always seek professional medical attention after first aid measures are provided. Eyes: Immediately flush eyes with excess water for 15 minutes, lifting lower and upper eyelids occasionally. Skin: Immediately flush skin with excess water for 15 minutes while removing contaminated clothing. Ingestion: Call Poison Control immediately. Rinse mouth with cold water. Give victim 1-2 cups of water or milk to drink. Induce vomiting immediately. Inhalation: Remove to fresh air. If not breathing, give artificial respiration.

Section 5: Fire Fighting Measures Noncombustible solution. When heated to decomposition, emits acrid fumes. 0 Protective equipment and precautions for firefighters: Use foam or dry chemical to extinguish fire. 1 0

Firefighters should wear full fire fighting turn-out gear and respiratory protection (SCBA). Cool container with water spray. Material is not sensitive to mechanical impact or static discharge.

Section 6: Accidental Release Measures Use personal protection recommended in Section 8. Isolate the hazard area and deny entry to unnecessary and unprotected personnel. Contain spill with sand or absorbent material and place in sealed bag or container for disposal. Ventilate and wash spill area after pickup is complete. See Section 13 for disposal information.

Section 7: Handling and Storage Green Handling: Use with adequate ventilation and do not breathe dust or vapor. Avoid contact with skin, eyes, or clothing. Wash

hands thoroughly after handling. Storage: Store in General Storage Area [Green Storage] with other items with no specific storage hazards. Store in a cool, dry,

well-ventilated, locked store room away from incompatible materials.

Section 8: Exposure Controls / Personal Protection Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash NIOSH-approved respirator with an dust cartridge. Exposure guidelines: Contains no materials with listed exposure limits.

Health 1 Fire Hazard 0 Reactivity 0





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MSDS # 778.00 Tris-Borate-EDTA Scholar Chemistry Section 9: Physical and Chemical Properties Molecular formula Mixture. Appearance Clear colorless liquid. Molecular weight N/A. Odor No odor. Specific Gravity 1.00 g/mL @ 20°C. Odor Threshold N/A. Vapor Density (air=1) N/A. Solubility Soluble in water . Melting Point N/A. Evaporation rate < 1 (Butyl acetate = 1). Boiling Point/Range N/A. Partition Coefficient N/A. (log POW). Vapor Pressure (20°C) N/A. pH N/A. Flash Point: N/A. LEL N/A. Autoignition Temp.: N/A. UEL N/A.

N/A = Not available or applicable

Section 10: Stability and Reactivity Stability: Stable under normal conditions of use and storage. Incompatibility: None known. Shelf life: Indefinite shelf life, store in a cool, dry environment.

Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, conjunctivitis. Skin: Redness, itching. Ingestion: Irritation and burning sensations of mouth and throat, nausea, vomiting and abdominal pain. Inhalation: Irritation of mucous membranes, coughing, wheezing, shortness of breath, Chronic Effects: No information found. Sensitization: none expected Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.

Section 12: Ecological Information Ecotoxicity (aquatic and terrestrial): Ecological impact has not been determined

Section 13: Disposal Considerations Check with all applicable local, regional, and national laws and regulations. Local regulations may be more stringent than regional or national regulations. Small amounts of this material may be suitable for sanitary sewer or trash disposal.

Section 14: Transport Information DOT Shipping Name: Not regulated by DOT. Canada TDG: Not regulated by TDG. DOT Hazard Class: Hazard Class: Identification Number: UN Number:

Section 15: Regulatory Information EINECS: Not listed . WHMIS Canada: Not WHMIS Controlled. TSCA: All components are listed or are exempt. California Proposition 65: Not listed. The product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS contains all the information required by the Controlled Products Regulations.

Section 16: Other Information Current Issue Date: January 10, 2012 Disclaimer: Scholar Chemistry and Columbus Chemical Industries, Inc., (“S&C”) believes that the information herein is factual but is not intended to be all inclusive. The information relates only to the specific material designated and does not relate to its use in combination with other materials or its use as to any particular process. Because safety standards and regulations are subject to change and because S&C has no continuing control over the material, those handling, storing or using the material should satisfy themselves that they have current information regarding the particular way the material is handled, stored or used and that the same is done in accordance with federal, state and local law. S&C makes no warranty, expressed or implied, including (without limitation) warranties with respect to the completeness or continuing accuracy of the information contained herein or with respect to fitness for any particular use.

ap® investigation #9 bio #9 v3.pdf...ward’s to adapt to the specifications outlined in ap biology...

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