Advanced Placement Biology - EDVOTEKEDVO-Kit # 283 Analysis of Cell Mitosis and DNA Extraction 3 table of Contents Page Experiment Components 3 Experiment Requirements 3 Background

®

Advanced Placement Biology

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283EDVO-Kit #

Analysis of Cell Mitosisand DNA Extraction

Storage: Store entire experiment

at room temperature

A.P. Biology

Lab 3

ExPEriMENt OBjECtiVE

The objective of this experiment is to demonstrate a staining procedure to identify the stages of Mitosis us-ing onion tissue. The mitotic phases will be compared and contrasted to the stages of Meiosis. Students will compare plant and animal Mitosis and isolate (spool) onion DNA.

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EDVO-Kit # 283 Analysis of Cell Mitosis and DNA Extraction

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table of Contents

Page

Experiment Components 3

Experiment Requirements 3

Background Information 4

Experiment Procedures

Experiment Overview 9

Part A. Preparation of Onion Root Tip Slides 10

Part B. Observation of Mitosis in the Whitefish Blastula 12

Part C Spooling of Onion DNA 13

Laboratory Extension Activity 14

Study Questions 15

Instructor's Guidelines

Notes to the Instructor 17

Pre-Lab Preparations 18

Study Questions and Answers 19

Material Safety Data Sheets 21

Advanced Placement (AP) Program is a regis-tered trademark of the College Entrance Exami-nation Board. These laboratory materials have been prepared by EDVOTEK, Inc. which bears sole responsibility for their contents.


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3A Onion DNA Extraction Buffer B 0.5% Toluidine Blue O Stain 15 ml Conical Test Tubes Gel Loading Solution, 10x

Experiment Components

• Microscopes• Microscope Slides and Cover slips• 10 Onion Bulbs• Pasteur Pipets and Bulbs (or glass rods for spooling)• 13 x 100 mm Test Tubes• Bunsen Burner and Striker• Razor Blades or Scalpel• 95% Ethanol or Isopropanol• 1 M HCl Acid• 5 ml Pipets• Computer with internet access• Cheesecloth or coffee filters (optional)

requirements

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

EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.

Storage: Store entire

experiment at room temperature.

This experiment is designed for 10 lab groups

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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 © 1991,1993,1996,1997, 1998, 1999, 2004, 2005, 2008 EDVOTEK, Inc., all rights reserved. EVT 080505AM

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centrosomes(with centriole pairs)

chromatin (duplicated)

aster

nucleolus

nuclearenvelope

plasma membrane

Background information

The growth and development of every organism depends on the precise replication of the genetic material during each cell division. It is remark-able when one considers that as individuals we have all arisen from the fertilization of a single egg with a single sperm. From this single cell we develop into unique individuals with highly differentiated tissue types. The instructions for the precise timing of development, growth and maturation are all contained within DNA, which is organized as nucleo-tides encoding specific genes, which are organized into chromosomes. Each cell contains this set of information. Differential gene expression is what accounts for the obvious differences between the various tissue types that make up nerves, skin, muscle and organs such as the kidneys, liver and spleen.

The cell cycle, the sequence of events that encompass the period between the completion of one cell division until the end of the next division, involves both division of the cell’s nucleus (karyokinesis) and division of the cytoplasm (cytokinesis) . There are two types of nuclear division: mitosis and meiosis. New body (somatic) cells are formed by mitosis. Each cell division produces two new daughter cells with the same num-ber and kind of chromosomes as the parent cell. The formation of male and female gametes in animal cells or spores in plant cells is by meiosis. Gametes and spores will have half the chromosome number of the parent cells.

interphase

Interphase, which begins when cell division ends and continues until the beginning of the next round of division, is organized into three phases. G1, is the first growth period of interphase. The nucleus and cell increase in size, and chromosomes are fully extended. The cell expends large amounts of energy in the synthesis of RNA and protein. During G1, the cell carries out normal functions specific to its type (i.e., nerve, liver, spleen). S, the next section of interphase, is marked by a dramatic

rise in DNA synthesis, and synthesis of histones that are major cellular proteins bound to DNA. The cell is preparing for the beginning of

mitosis. The chromosomes are becoming longitudinally doubled, with each chromosome consisting of two identical “chroma-

tids”. G2, the final segment, is marked by continued protein synthesis. A cell in interphase has a nucleus with one or more dark-stained nucleoli and a fine network of threads, the chromatin.

Figure 1: G2 Interphase

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chromosome with 2 sister chromatids

centromere

early mitoticspindle

Figure 2: Prophase

Figure 3: Metaphase

Mitosis

Mitosis is the next phase of the cell cycle. It is the process of coordinated chromosome replication prior to cell division. It is essentially the same whether considering a simple plant or a highly evolved organism, such as a human being. The major function of mitosis is to accurately and precisely replicate genetic information, or chromosomes, so each daugh-ter cell contains the same information. The enzymatic complex, a DNA polymerase, accomplishes this task with an average of less than one error, or one base pair change per 1 X 109, nucleotides synthesized. The human genome contains approximately 3.3 X 109 base pairs, so less than 3 errors would occur during a typical cell division.

The process of mitosis is an ongoing event that can be segmented into several identifiable stages. During the mitotic phase, a unique compli-ment of genes are activated. These genes encode proteins which act only transiently during mitosis and are absent from other phases of the cell cycle. In order, these stages are: prophase, metaphase, anaphase, and telophase. Cytokinesis, the actual process of cell division, occurs during telophase. In plants such as the onion, this is seen as the formation of the cell plate between the two daughter cells.

Prophase

In prophase, dramatic changes begin to occur within the nucleus of the cell. Chromosomes become thicker, shorter, and easily visible when stained under the light microscope. Two “sister chromatids” join near their middle at a structure called the centromere. The nucleolus, the site of active rRNA synthesis, and the nuclear membrane disappears. The mitotic apparatus, the spindle, begins to organize within the cell. Mi-crotubules are slender rods of protein responsible for pulling replicated chromosomes towards each half of the cell. In animals, the centrosome splits into two centrioles which move to the poles of the cell. The spindle seems to radiate from these two centrioles.

Metaphase

During this period, chromosomes become aligned at midpoint or equator between poles of the cell and are at their thickest and shortest structure. They are easily identified as two longitudinally double sister chromatids. In animals and plants, chromatids are connected (at their centromeres) to the spindle apparatus, which has formed between the two centrioles located at the poles of the cell. In many plants, the centrioles are absent. The spindle is still present, however, and the plant chromosomes are simi-larly attached to the spindle microtubular fibers.




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nucleolusforming

cleavagefurrow

nuclearenvelope forming

Figure 5: Telophase andCytokinesis

Anaphase

In this short phase, sister chromatids begin to separate and migrate to the poles. Once the two chromatids separate, each is called a chromo-some. For humans, with a diploid number of 46 chromosomes, there will be 46 chromosomes moving toward each pole. Onions have 16 diploid chromosomes and, therefore 16 chromosomes move to each pole. Dur-ing anaphase there is a quantitative, equal segregation of the diploid number of chromosomes into two developing nuclei at the poles of the anaphase cell.

telophase and Cytokinesis

The final mitotic phase of the cell cycle is recognized by the formation of two new nuclei encompassing the daughter chromosome at the cell poles. The mitotic apparatus disappears and chromosomes begin to lengthen as they unwind. Cytokinesis, formation of a new cell mem-

brane, occurs midway between the daughter nuclei. In animals, there is the formation of the indented cleavage furrow. In plants, such as the onion root tip cells, this is seen as the formation of a cell plate, dividing the original cell into two (presumably equivalent) daughter cells. Cells now enter the G1, stage of interphase in the cell cycle and the process begins anew.

Meiosis

Meiosis is a specialized type of cell division sharing many features with mitosis. The main difference is that meio-

sis involves two successive nuclear divisions that produces four haploid cells. Each gamete, or sex cell, contains half the

number of chromosomes. In humans, each gamete contains 23 chromosomes. Fertilization of an egg by a sperm, each containing

23 chromosomes, restores the diploid number of 46 chromosomes. Meio-sis consists of two rounds of cell division, Meiosis i and Meiosis ii, each with its own prophase, metaphase, anaphase and telophase.

In animals, the gametes, sperm and egg of animals are generally formed directly from diploid tissue rather than from a haploid gametophyte generation in plants such as corn. In animals, the egg and sperm join to form the diploid zygote which develops into a mature adult. In plants, one of the male gametes from the pollen (formed in the stamens) unites with the female gamete in the pistil to form the fertilized diploid zygote. The other male gamete combines with the diploid endosperm nucleus to form a triploid endosperm tissue. Both are in the corn seed. For a more thorough discussion of reproduction in plants and animals, consult your textbooks.

Figure 4: Anaphase

daughter chromosomes



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interphase

Chromosomes would exist as chromatin at this stage and not as visible struc-tures. DNA synthesis would occur resulting in each strand made up of two strands or chromatids, joined to each other at the centromere.

MEiOtiC DiViSiON #1

Prophase i

The chromosomes begin to shorten and thicken. In some plants, they ap-pear to aggregate together on one side of the nucleus. In animals, they may appear to orient with one end nearest the nuclear membrane adjacent to the centriole. the first major difference between mitosis and meiosis is that homologous pairs of chromosomes come together or synapse. A tetrad consisting of four chromatids is the result. This complex allows for “crossing over” to occur between the homologous pairs of chromosomes. The point of crossing over appears as an X shaped structure, called the chiasma, (chiasma-ta, plural). During the formation of the chiasmata, there is a crossing over, or genetic exchange, between homologous chromosomes. There is an enzyme catalyzed breakage and repair of the synapsed chromosomes. Crossing over is very important because it leads to an increase in genetic randomness and species/genetic diversity. The last step is the ending of chiasmata formation, disappearance of the nucleolus and nuclear membrane, and formation of the mitotic spindle.

Metaphase i

The synapsed homologous pairs of chromosomes arrive at the midpoint, or equator, between poles. The synapsed pairs orient such that one member of each pair faces the opposite pole of the cell, with the 23 pairs of chromo-somes arranged entirely in random fashion. There is no tendency for one member of the pair to face one of the poles. This random assortment also contributes heavily to genetic diversity within a species.

Anaphase i

The pairs of homologous chromosomes, each longitudinally double (tetrads), begin to separate and migrate to the cell poles. As contrasted to mitosis, en-tire chromosomes, versus the sister chromatids, move to each pole. this is the second major difference between mitosis and meiosis. Each pole randomly receives either the maternal or paternal chromosome of each homologous pair. Therefore, there is an exact halving of the diploid chromosome number during Anaphase I stage of meiosis.



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telophase i

The chromosomes arrive at the poles of the cell at the beginning of this phase. The nuclear membrane forms and the nucleolus begins to reorganize. Cytokines is, physical cell division, occurs during this phase, although not in all animal or plant species. In corn, there is a physical separation during this stage. In the plant Trillium, Telophase I appears to be skipped entirely.

Interphase II (Interkinesis). How much time spent in this phase depends on the type of organism, the formation of new nuclear envelopes, and the amount of chromosomal uncoiling. A third major difference be-tween mitosis and meiosis is that DNA replication does not occur during interkinesis.

MEiOtiC DiViSiON #2

In order to reduce the amount of DNA to half, a second meiotic division is necessary to separate the chromatids of the chromosomes in the two daughter cells formed in Meiosis #1.

Prophase i

This phase resembles mitotic prophase except the chromosomes do not dramatically shorten. The nucleolus, the site of active rRNA synthesis, disappears. The nuclear membrane also disappears and the mitotic ap-paratus, the spindle, begins to organize within the cell.

Metaphase ii

The monoploid number of chromosomes organizes at the midpoint (equator) between the poles. Each chromosome is composed of two sister chromatids.

Anaphase ii

The sister chromatids begin to separate and migrate to the poles as in mitosis. This stage ends when they are at the poles. Each chromatid has its own separate centromere region now, and it is called a chromosome.

telophase ii

The chromosomes begin to lengthen, the nucleus reforms, and the nucle-olus reorganizes. Cytokinesis occurs and the final result of meiosis is four cells each containing the haploid chromosome number of chromosomes.



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Experiment Overview

ExPEriMENt OBjECtiVES:

Students Will: 1. Learn a staining procedure to identify the stages of mitosis using

onion tissue. 2. Calculate the relative duration of the cell cycle stages. 3. Compare and contrast plant and animal mitosis. 4. Compare and contrast the stages of mitosis to the stages of meiosis. 5. Isolate (spool) onion DNA from the nucleus.

WOrKiNG HYPOtHESiS

If somatic cell division involves a sequence of biological events from the completion of one cell division to the end of the next and results in two new daughter cells with the same number and kind of chromosomes as the par-ent cell, then germ cell formation (male and female gametes) will undergo a similar sequence of biological events twice from the beginning to the completion of two successive nuclear divisions and result in four new daugh-ter cells each with half the number of chromosomes with genetic variations.

MAtEriALS FOr tHE ExPEriMENt

Each Lab Group should have the following materi-als for Parts A, B, & C:

• Microscope• Slides and cover slips• Scalpel• Toothpicks• Onion root tip• Bottle 1N HCl• Slide holder• Bunsen burner• Bottle 0.5% Toluidine Blue O Stain• Paper Towels

LABOrAtOrY SAFEtY

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

OBSErViNG MitOSiS iN PLANt CELLS

The region of cells with the largest percentage of cells undergoing mitosis on the onion slide is located im-mediately above the root cap cells in a region known as the apical meristem. The region immediately above the meristem is the region of elongation. Most of the cells will be in interphase in this region. Above this region is the region of maturation. This is where cells differentiate to become xylem, phloem, and other tis-sues. It is also the region where root hairs develop.




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Part A. Preparation of Onion root tip Slides

You will prepare slides containing stained onion root tip squash sec-tions, which will allow you to identify relevant stages of mitosis. Your instructor has prepared freshly rooted onions for use in this procedure. Remember it is the tips of newly emerging roots that contain the highest proportion of cells undergoing mitosis.

1. Obtain the newly rooted onion from your instructor. Cut off the first 1-3mm of the root tip using a straight edged razor blade or a scalpel and transfer to a clean microscope slide.

2. Cover the root tip cross section with 2-3 drops of 1N HCl. Using a slide holder (forceps or clothespins can be substituted), pass slide over Bunsen burner flame for 3-5 seconds.

3. Remove slide from flame and allow to cool. Blot up excess HCl care-fully using a paper towel.

4. Cover the root tip with 3-4 drops of 0.5% Toluidine Blue O Stain, Component B.

5. Pass the slide carefully above the Bunsen burner flame 2 or 3 times. (The stain should not boil.) Allow the slide to cool for 1 minute.

6. Blot up excess stain from periphery of root tip.

7. Add 1 drop of 0.5% Toluidine Blue O Stain to root tip and cover with a cover slip.

8. Place the slide between a folded paper towel and place onto your lab bench.

9. Carefully and gently use an eraser or rubber stopper to apply pres-sure on top of the coverslip. This will spread out the stained onion root tip for visualization.

10. As with all attempts to visualize material under the microscope you should begin at the lowest lens power that will visualize the objects of interest. Then shift to higher lens powers for further viewing.

11. Sketch the phases you observe. You should be able to identify all of the mitotic stages including: prophase, metaphase, anaphase, telo-phase and the nondividing stage, interphase. Record the number of cells in each stage.

12. Record the number of cells in each stage. Count at least three full fields of view. You should have counted over 200 cells.




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13. Record your data in table i, below.

14. Calculate the percentage of cells in each phase and record in the table below. Estimate time spent in each stage by the following calculation:

Number of cells in stage total # cells in all stages

15. Calculate the amount of time spent in each phase of the cell cycle from the percentage of cells in that stage. On the average, it takes 1,440 minutes (24 hours) for onion root tip cells to complete the cell cycle.

% of cells in stage x 1��0 minutes = _______ minutes of cell cycle spent in stage.

total Cells Counted

Table INumber of Cells Percent of total time in Cells Counted Each Stage

Field 1 Field 2 Field 3 total

Part A. Preparation of Onion root tip Slides

interphase

Prophase

Metaphase

Anaphase

telophase



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Part B. Observation of Mitosis in the Whitefish Blastula

The whitefish blastula digital images provide the ability to visualize stages of mitosis in animal cells. The blastula represents one of the early development stages following fertilization of the whitefish egg. All developing embryos pass through this stage. Cells which give rise to the varied tissue types found in the adult whitefish are forming during this development stage. The description of mitosis in the Background Information section of this experiment is general and applies equally to mitosis in animal and plant cells. Refer to that description to help in identifying the stages of mitosis in animal cells.

1. Access the internet and using a search engine (e.g. Google), type "whitefish blastula mitosis". Look for images of the mitotic stages.

2. Draw examples of the mitotic stages which you observe.

3. Note and list any differences you see between the stages of animal mitosis and the stages of plant mitosis. Discuss with the class.

One clear difference is the deep cleavage furrow, a characteristic of cyto-kinesis in animal cells. Compare this to the cell plate in plant cytokinesis.



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Part C. Spooling of Onion DNA

Figure 10

Figure 9

The chromosomes you have just seen are composed of Deoxyribonucleic Acid (DNA). The entire genetic information required for the growth and reproduction of the onion is encoded within this DNA in the form of genes. In this procedure, you will isolate DNA from the onion and spool the DNA from solution.

1. Carefully slice a 5 x 5 x 5 mm cube (1.5 gm) from the main body of the onion (not the root tip), and place into a 15 ml conical test tube.

2. Add 2.0 ml of Onion DNA Extraction Buffer to the tube and mince up the onion cube with the tip of a pencil or pen. This will release the cellular contents including the onion DNA from the onion cells.

3. Carefully remove the onion extraction buffer solution from the test tube by pipetting or decanting. Place into a fresh 13 x 100 mm test tube. Take care to minimize carry over of the onion tissue. Alterna-tively, you may filter the contents of the tube through several layers of cheesecloth into a clean tube (squeeze out the liquid).

See Figure � at left.

4. Carefully overlay the aqueous solution with 2.0 ml of 95% cold Iso-propanol or cold 95% Ethanol. Alternatively, you can use 4.0 ml 70% Isopropanol. Allow the alcohol to flow slowly down the side of the test tube.

Since the density of alcohol is less than that of the aqueous phase, it will float on top. Two phases can be observed.

5. Place a glass Pasteur pipet or glass rod into the test tube and begin to spin with your fingers. Rotate the pipet enough to mix the two phases. The DNA, which is insoluble in alcohol, will begin to spool (wrap) around the glass.

6. Remove the pipet from the test tube after spooling for several min-utes.

The DNA will appear as a viscous material adhering to the pipet. The DNA may be grayish in color if a pencil was used to mince the onion tissue. See Figure 10 at left.

7. Rinse the spooled DNA with 95% ETOH (ethanol) to remove excess proteins. This will help to expedite the rehydration process.

8. Resuspend the DNA in 2 ml of distilled or deionized water in a fresh 13 x 100 mm test tube.

8. Cover the test tube with saran wrap or parafilm and allow the test tube to stand overnight at room temperature.



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The resuspended DNA can be electrophoresed on an agarose gel and visu-alized by staining. To prepare a sample for electrophoresis:

• Transfer 0.1 ml of DNA solution to a fresh 13 x 100 mm test tube. Do not worry about the undissolved DNA.

• With a Pasteur pipet, add 2 drops of 10 X Gel Loading Solution. Mix.

• The sample is ready to be applied to a 0.8% agarose gel.

• Use conditions recommended by the manufacturer for the electro-phoretic separation.

• To visualize DNA after electrophoresis, stain with InstaStain® Methy-lene Blue.

Laboratory Extension Activity



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Study Questions

1. What is the significance of the “S” phase of Interphase of cell divi-sion?

2. What are the specific differences between animal and plant karyoki-nesis and cytokinesis?

3. Based on the number of cells you found in each stage of mitosis in the onion root tip, what stage of mitosis is the longest?

4. List and explain the 3 major differences between mitosis and meiosis during Prophase I, Anaphase I and Interphase II.

5. Compare mitosis and meiosis by completing the following table:

6. What is the major difference between Meiosis I and Meiosis II?

7. When does crossing over usually occur? Why is this significant?

8. How does the frequency of crossing over relate to the distance be-tween genes?

9. Why is it important to know the percentage of recombinants in off-spring?

10. What is a map unit?

11. Why does DNA spool onto the glass?

12. Is DNA soluble in 95% alcohol?

Mitosis Meiosis

Parent Cell Chromosome Number

Number of DNA Replications

Number of Cell Divisions

Number of Daughter Cells Produced

Daughter Cell Chromosome Number

Major Significance



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Notes:



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1�EDVO-Kit # 283 Analysis of Cell Mitosis and DNA Extraction

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technical ServiceDepartment

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

Please have the following information:

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OVErViEW OF LABOrAtOrY iNVEStiGAtiONS

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EDVOTEK® experiments have been designed to provide students the op-portunity to learn very important concepts and techniques used by scientists in laboratories conducting biotechnology research. Some of the experimen-tal procedures may have been modified or adapted to minimize equipment requirements and to emphasize safety in the classroom, but do not com-promise the educational experience for the student. The experiments have been tested repeatedly to maximize a successful transition from the labora-tory to the classroom setting. Furthermore, the experiments allow teach-ers and students the flexibility to further modify and adapt procedures for laboratory extensions or alternative inquiry-based investigations.

OrGANiziNG AND iMPLEMENtiNG tHE ExPEriMENt

Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances.

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Notes to the instructor







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Pre-Lab Preparations

PArt A: PrEPArAtiONS FOr ONiON rOOt tiP SLiDES Approximately �8 hours before the laboratory:

Preparation of Onion root tips

• Rinse the onions under water to remove excess dirt.

• Carefully and gently slice approximately 1 cm above the root end with a knife or razor blade. Save onion tissue for Part C.

• Wrap each onion in a wet paper towel and place into a sealable plastic bag. Place the bag into a desk drawer and close the drawer to exclude light.

• In the next 24 - 48 hours, each onion will have a number of newly emerging root tips.

Preparation of 1N (1M) HCl

• Add 2 ml of concentrated Hydrochloric Acid (12N) to 22 ml distilled or deionized water. Mix.

Caution: Always gently add concentrated acid to water

• Each lab group requires 2 ml.

PArt B: OBSErVAtiON OF MitOSiS StAGES

• Access the internet and use a search engine (e.g. Google) to find digital images of whitefish mitosis stages.

PArt C. PrEPArAtiONS FOr SPOOLiNG OF ONiON DNA

• Place 30 ml of 95-100% Isopropanol or 60 ml of 70% Rubbing alco-hol on ice or in the refrigerator.

Always gently add concentrated acid to water



Please refer to the kit insert for the Answers to

Study Questions

Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.

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

Section IManufacturer's Name

Section II - Hazardous Ingredients/Identify Information

Emergency Telephone Number

Telephone Number for information

Date Prepared

Signature of Preparer (optional)

Address (Number, Street, City, State, Zip Code)EDVOTEK, Inc.

14676 Rothgeb DriveRockville, MD 20850

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

Other Limits Recommended % (Optional)

(301) 251-5990

(301) 251-5990

Boiling Point

Section III - Physical/Chemical Characteristics

Unusual Fire and Explosion Hazards

Special Fire Fighting Procedures

Vapor Pressure (mm Hg.)

Vapor Density (AIR = 1)

Solubility in Water

Appearance and Odor

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

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)

Specific Gravity (H 0 = 1) 2

Onion DNA Extraction Buffer

CAS # 7631-98-3N-lauroylsarcosine sodiumsarcosine, N-dodecyl-, sodium salt

98°C

No data

No data

1.91

No data

No data

soluble

Clear liquid, no odor

Water spray, CO2, Dry chemical powder or Polymer foam

Wear SCBA and protective clothing to prevent contact with skin and eyes

None

StabilitySection V - Reactivity Data

Unstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

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

Other

Stable

Hazardous Polymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

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

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

X

Iron, steel, aluminum, or copper

X

Toxicological properties have not been thoroughly investigated

no data

Treat supportively and symptomatically

Mop up with absorbent material. Dispose of properly

Bury in landfill site approved for disposal of chemical and hazardous waste. Observe all federal, state, and local laws.

Avoid contact. Do not store in contact with iron, steel, aluminum or copper

None

rubber boots

Avoid contact

Toxic fumes of carbon monoxide, carbon dioxide, nitrogen oxides

None

Yes Yes Yes

Can cause coughing, wheezing, respiratory distress, and diarrhea. Skin/eye irritation

No data

NIOSH/MSHA approved respirator

No NoneYes None

Chemical resistant Safety goggles

Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.

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

Section IManufacturer's Name

Section II - Hazardous Ingredients/Identify Information

Emergency Telephone Number

Telephone Number for information

Date Prepared

Signature of Preparer (optional)

Address (Number, Street, City, State, Zip Code)EDVOTEK, Inc.

14676 Rothgeb DriveRockville, MD 20850

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

Other Limits Recommended % (Optional)

(301) 251-5990

(301) 251-5990

Boiling Point

Section III - Physical/Chemical Characteristics

Unusual Fire and Explosion Hazards

Special Fire Fighting Procedures

Vapor Pressure (mm Hg.)

Vapor Density (AIR = 1)

Solubility in Water

Appearance and Odor

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

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)

Specific Gravity (H 0 = 1) 2

B/283 Toluidine Blue O Stain

CAS # 92-31-9This product contains no hazardous components

No Data

No Data

No Data

No Data

N/A

No Data

Highly soluble

Dark blue solution

Water spray, CO2, Dry chemical powder or appropriate foam

Wear SCBA and protective clothing to prevent contact with skin and eyes

Emits toxic fumes under fire conditions.

StabilitySection V - Reactivity Data

Unstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

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

Other

Stable

Hazardous Polymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

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

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

If swallowed rinse mouth with water. Skin contact, flush with water. If inhaled move to fresh air.

Mop up with absorbent material. Dispose of properly

Mix with combustible solvent and burn in chemical incinerator equipped with afterburnerand scrubber. Observe all federal, state and local laws.

Wear protective equipment

avoid contact and inhalation

NIOSH/MSHA approved respirator

No No

Yes None

Chemical resistant Safety goggles

X

No data

X

May cause irritation

No data

No data

No data

None

Avoid contact and inhalation

Toxic fumes of Carbon Monoxide, Carbon Dioxide, Nitrogen Oxides, Sulfuroxides, HCL gas

Yes Yes Yes

Documents

Advanced Placement Biology - EDVOTEKEDVO-Kit # 283 Analysis of Cell Mitosis and DNA Extraction 3 table of Contents Page Experiment Components 3 Experiment Requirements 3 Background