Lab Genetics - Labwork Manual.pdf

VIETNAM NATIONAL UNIVERSITY HCMC INTERNATIONAL UNIVERSITY SCHOOL OF BIOTECHNOLOGY

GENETICS LABORATORY MANUAL

Student name:................................... ID: .................................................... Year: ...............................................

HCMC-2015

CONTENTS

LABORATORY SAFETY GUIDELINES .......................................................2 LAB 1: Culturing and Experimental Mating of Drosophila melanogaster .......... 6 LAB 2: Mitosis ................................................................................................... 11 LAB 3: Colchicine-induced Polyploidy ............................................................. 14 LAB 4: Meiosis................................................................................................... 16 LAB 5: Polytene Chromosome of Drosophila melanogaster ............................ 20 LAB 6: Extraction of DNA from plant cells ...................................................... 24 APPENDIX - RECIPES ................................................................................... 27

HCMC-2015

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INTRODUCTION TO GENETICS LABWORK This lab is designed to give students an introduction to major organisms used for studying genetics. We will explore the classical and modern techniques. In this practical the students will perform technique in culturing fruit flies in vials, distinguish male and female fruit flies, and identify various stages in the life cycle of D. melanogaster. To understand how the genetic material passed between generations, how gene expression may be regulated, and how mutation can occur. The students will perform the practical in onion root tip samples and garlic chive flower samples; observe the steps of cell division. Practical provide to gene map study also carry out in this lab through the structure of the polytene chromosome.

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LABORATORY SAFETY GUIDELINES A. GENERAL LABORATORY SAFETY 1. Work carefully and cautiously in the laboratory, using common sense and good judgment at all times. 2. EATING. DRINKING AND SMOKING ARE PROHIBITED in the laboratory and in the laboratory space of a combined lecture/laboratory room. 3. Long hair must be tied back during laboratory sessions. 4. Open toed shoes are prohibited. 5. No sleeveless tops are permitted. Lab coats must be worn. 6. Identify the location of all exits from the laboratory and from the building. 7. Be familiar with the location and proper use of fire extinguishers, fire blankets, first aid kits, spill response kits, and eye wash stations in the laboratory. 8. Report all injuries, spills, breakage of glass or other items, unsafe conditions, and accidents of any kind, no matter how minor, to the instructor immediately. 9. Keep sinks free of paper or any debris that could interfere with drainage. 10. Lab tables must be clear of all items that are not necessary for the lab exercise. 11. Wash hands and the lab tables with the appropriate cleaning agents before and after every laboratory session. B. OPEN FLAMES - FIRE HAZARD 1. Identify and be familiar with the use of dry chemical fire extinguishers that are located in the hallways and laboratory rooms. 2. Flames are only to be used under the supervision of the instructor. C. SHARP OBJECTS AND BROKEN GLASS 1. Pointed dissection probes, scalpels, razor blades, scissors, and microtome knives must be used with great care, and placed in a safe position when not in use. 2

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2. Containers designated for the disposal of sharps (scalpel blades, razor blades, needles; dissection pins, etc.) and containers designated for broken glass are present in the laboratory. Never dispose of any sharp object in the regular trash containers. 3. Report all cuts, no matter how minor, to the instructor. 4. All biology labs and the biology preparation room (702) house a first aid kit containing antiseptics, bandages, Band-Aids and gloves to care for minor cuts. 5. Do not touch broken glass with bare hands. Put on gloves and use a broom and dustpan to clean up glass. Dispose of ALL broken glass in the specific container marked for glass. Do not place broken glass in the regular trash. 6. When cutting with a scalpel or other sharp instrument, forceps may be used to help hold the specimen. Never use fingers to hold a part of the specimen while cutting. 7. Scalpels and other sharp instruments are only to be used to make cuts in the specimen, never as a probe or a pointer. D. INSTRUMENTS AND EQUIPMENT Care must be used when handling any equipment in the laboratory. Students are responsible for being familiar with and following correct safety practices for all instruments and equipment used in the laboratory. Microscope Handling 1. Microscopes must be carried upright, with one hand supporting the arm of the microscope and the other hand supporting the base. Nothing else should be carried at the same time. 2. Microscope must be positioned safely on the table, NOT near the edge. 3. After plugging the microscope into the electrical outlet, the cord should be draped carefully up onto the table and never allowed to dangle dangerously to the floor. 4. The coarse adjustment must NEVER be used to focus a specimen when the 40x or oil immersion lens is in place. 3

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5. When finished with the microscope, the cord should be carefully wrapped around the microscope before returning it to instructor. 6. The microscope must be placed upright and in the table near the cabinet. 7. All prepared microscope glass slides are to be returned to their appropriate slide trays; wet mount preparations are to be disposed of properly. 8. Malfunctioning microscopes should be reported to the instructor. Hot Plates and Water Baths 1. The instructor will regulate the temperature of hot plates and water baths with a thermometer. 2. This equipment must be placed in a safe place. 3. Use insulated gloves or tongs to move beakers or test tubes in and out of the water baths. 4. Use care when working near hot plates and water baths, as they may still be hot even after being turned off. E. ADDITIONAL LABORATORY SAFETY FOR THE GENETICS LAB 1. The Drosophila melanogaster that are used for the lab experiments are not fit for the natural environment and are mutations of the wild form of this animal. They will not be released into the open air but euthanized in mineral oil. 2. Fly nap (triethylamine and ethanol) is to be used only as directed by the instructor and following the manufacturers recommendations. 3. DNA recombination experiments will only include DNA from the bacteria E. coli and it's plasmids. These recombinant organisms will be disposed of in biohazard receptacles. 4. E. coli strains used in the laboratory are not pathogenic and cannot survive in the adult large intestine. 5. Protective goggles will be used when viewing gels illuminated with ultraviolet light. 6. Gloves will be worn during molecular experiments and will be changed if there is a tear or spill. 4

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7. When agarose gels are stained with ethidium bromide only the instructor will handle the gel and the staining solution. Ethidium bromide is a mutagen by the Ames test and suspected carcinogen.

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LAB 1: CULTURING AND EXPERIMENTAL MATING OF DROSOPHILA MELANOGASTER I. Introduction Drosophila melanogaster is a cosmopolitan species, which can be found all over the world, including in your home if you have overripe fruits in the summer. Fruit fly has been widely used for genetic investigation because it has many characteristics of a good experimental model, including the short life cycle, the ease of culturing, the high reproductive rate, and the small chromosome number. General structure of the adult fruit fly Drosophila’s body has three main parts: the head, the thorax, and the abdomen. The major structures of the head are two big compound eyes, two antennas, a mouth. The thorax has six legs, two wings, and two halteres, which are small, club-shaped structures behind the wings that ensuring the fly balance when flying. On the dorsal surface of the thorax, there are a number of long dark bristles. It is not difficult to distinguish a female from a male fly. The male is usually smaller than the female. In addition, the female has more pointed abdomen, which is stripped rather than back-tipped (Fig. 1.1). Other features that can be used to determine the sex of the adult D. melanogaster is shown in Table 1.1. Table 1.1 Features for sex determination of fruit fly. Characteristics Overall size

Male Generally smaller the female

Female Generally larger than male

Size of abdomen

Smaller than female’s

Larger – due to distention with maturing eggs

Shape of abdomen

Narrower than female

Broader

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Color of tip of

Tip rounded (blunt)

Tip is pointed

Solid black dorsally

Not so

Small, dark spot on ventral tip of

Absent

abdomen Penis

abdomen. This, with dorsal black area, makes tip of abdomen appear entirely black Number of

5

7

Present; about 10 dark, short, curved

Absent

abdominal segments visible Sex comb#

bristles (tufts) on the front legs #

Sex comb is special bristle found on the front legs of the male fruit fly. It has no sexual

function and can be observed at the 100-fold magnification.

Life cycle Drosophila life cycle consists of the following four main stages (Fig. 1.1) • Eggs. Eggs are laid by the mother on the food and take about one day to hatch. They are small and translucent with two “ears” sticking out. • Larva. Larvae are maggots, which crawl through the food in jerky motion, eating as they go. The larvae go through three molts: they are hatched from the egg as small, “the first instar larvae”. Then after a day they molt to become larger, “the second instar larvae”. After two days, “the third instar-larvae” climb up the wall of the vial, glue themselves to the glass, invert their spiracles (breathing tubes), and settle down as pupae. • Pupa. Pupae are the cocoons in which the larvae metamorphose into adults. The larval cuticle becomes a shell, its muscle melt away, and the new adult exoskeleton as well as musculature form inside. The pupal 7

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stage lasts five days. In the last day, you can see the red eyes and the dark wings forming inside. • Adult. The adult emerges from the pupal case as a white, elongated fly, whose wings are stilled folded up. After about an hour, the wings will expand and the body will take its normal shape and coloration. The adults become sexually mature after 8-10 hours. After this time, the males chase the females about in an endless quest for mating. Flies can live up to three months, but they are pretty decrepit after 6 weeks or so.

Figure 1.1 Life cycle of Drosophila melanogaster The length of life cycle and each stage vary according to the temperature. Average lengths of developmental periods are as follows. Temperature

10oC

15oC

20oC

25oC

28oC

30oC

31oC

Egg-Larval

57.0

17.3

8.1

5.0

4.2

4.0

5.0

Pupae-Adult

13.7

13.3

6.5

4.2

3.4

3.4

3.4

Total (days)

70.7

30.6

14.6

9.2

7.6

7.4

8.4 8

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II. Objectives Upon completion of this investigation, the student should be able to • Culture fruit flies in vials, • Distinguish male and female fruit flies, and • Identify various stages in the life cycle of D. Melanogaster. III. Materials Saccharose

Forceps

Propionic acid

Small pain brushs

Dry baking yeast

Petri dish

Agar

Etherizer flask

Water

Funnel

Ether

Cotton mesh

Culture tube

Sterile tissue papers

250 ml beaker

Stereo dissecting microscope

IV. Procedure A. Preparing media Each group of students will prepare 4 medium vials for culturing of the fruit fly 1.

Boil 0.75 g agar in 50 ml of water until the agar completely dissolve

2.

Add 2.5 g of saccharose and 2.5 g of dry baking yeast, and then boil the mixture for 1 minute with occasional stirring.

3.

Add 250 µl of propionic acid and stir well.

4.

Pour the medium into the sterile vials to the depth of about 2 cm. Take care to prevent the medium from coming into contact with the neck of the vial.

5.

Close the vial of the cotton plug, and leave the medium to cool to room temperature.

6.

Before put the flies into the tubes for culturing, dry the inner wall of the tube with sterile tissue papers.

B. Handling flies

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1.

Put some drops of ether on a piece of cotton mesh inside the funnel that is put on top of the etherizer flask.

2.

Strike the base of the culture tube lightly on the palm of the hand so the flies will drop to the bottom.

3.

Remove the plug and quickly invert the tube over the funnel.

4.

Expose the flies to ether for about 1-2 minutes after they stop moving. Overetherization will kill the flies.

5.

Transfer some etherized flies to a dry petri dish.

6.

Examine the flies under the dissecting microscope with the light source shining from above. Use a small paintbrush to move the flies about on the petri dish.

7.

Divide the flies in to two groups based on their sex.

8.

Open the plug of the culture tube, keep the tube horizontally and gently transfer three female and three male flies into the tube.

9.

Wait until the flies revive before turn the tube vertically otherwise the flies come into contact with the moist medium, wetting their wings and unable to fly out of the medium.

10. Keep the tubes in cool place for one week or more. 11. Observe the tube to identify different stages in the life cycle of Drosophila. The third instar larvae will be used for practical 6. V. Report 1. Record the following morphological characteristic of flies that you observe: • Body color: ......................................................... • Eye color: ............................................................ • Wing shape: ........................................................ • Presence of antenna: ........................................... 2.

Record morphological states of the Drosophila that you can observe in the tube after one and two weeks.

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LAB 2: MITOSIS I. Introduction Mitotic cell division is the type of cell division that results in the formation of two genetically identical daughter cells from a single mother cell. Before entering mitotic cell division, cell spends long time in the interphase to acquire or synthesize materials that are essential for cell division and to replicate its DNA. Mitotic cell division consists of two consecutive processes: mitosis (nuclear division) and cytokinesis (cytoplasmic division). During mitosis, duplicated chromosomes, each consists of two sister chromatids, condense into visible threadlike structures then equally separated into the opposite end of the cell. Following mitosis, nucleus is formed in each daughter cell and the cytoplasm is divided into each daughter cells through the process of cytokinesis. Mitosis can be divided into four phases: prophase, metaphase, anaphase and telophase (Fig. 2.1): During prophase, the chromosomes condense and the spindle microtubles form and attach to the chromosomes. The site on chromosome that binds to the spindle is called kinetochore. Kinetochores of sister chromatids are tethered to the spindles that are originated from opposite pole of the cell. Nuclear envelope breaks down and the nucleolus disappears. During metaphase, the chromosomes line up in a single plane (the metaphase plate) along the equator of the cell. During anaphase, chromatids of each chromosome separate and move to the opposite poles of the cell. The chromatids form V-liked structures with the centromeres pointing toward the respective poles. Telophase starts when the chromosomes reach the poles. During this phase, the chromosomes relax, the nuclear envelope is re-synthesized around both groups of chromosomes, the spindle apparatus is dismantled, and the nucleoli reappear. Cytokinesis usually occur during telophase, separating the two nuclei into separate cells. 11

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Interphase

Prophase

Metaphase

Anaphase

Telophase Figure 2.1 Chromosomes’ behavior during mitotic cell division

II. Objectives Upon completion of this investigation, the student should be able to • Outline the procedure to prepare acetocarmin squashes of onion root tips, and • Describe in chronological order the main events of mitosis in onion root tip.

III. Materials Onion bulbs

Watch glasses

1M HCl

Forceps

Distilled water

Scissors

70% & 90% ethanol

Sand

Acetocarmin stain Carnoy’s solution Microscope Microscope slides & coverslips

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IV. Procedure Before the practical, your instructor put the onion bulbs on top on moisten sand until their roots grow to about 5 cm in length. Approximately 1 cm of the root tip was collected and soaked in Carnoy’s solution for 12 h. Next the roots were washed twice with 90% ethanol for 10 minutes each time. They were then stored in 70% ethanol until use. Each student will prepare acetocarmin-stained squash of onion roots by the following procedure: 1.

Use a forceps to transfer some onion roots into a watch glass.

2.

Wash the roots with water 2-3 times to remove the ethanol.

3.

Remove all water then soak the roots in several drops of 1M HCl for 10-15 minutes to soften them. After that, wash the roots 2-3 times with water.

4.

Soak the roots in acetocarmine stain for 15-20 minutes.

5.

Put one drop of water at the middle of a microscopic slide then transfer one stained onion root into the water drop.

6.

Cover the root with a coverslip. Avoid creating bubble under the coverslip.

7.

Gently apply pressure with your thumb over the cover glass to squash the root tip into a thin layer.

8.

Examine the specimen under low power (10X objective) to identify the meristem area, where dividing cells are located. Then use higher power objective lens to carefully examine cells in various mitotic stages.

V. Report Draw and clearly label cells in various mitotic phases that you observe.

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LAB 3: COLCICINE-INDUCED POLYPLOIDY I. Introduction Polyploidy is referred to cells or organisms that contain cells with more than two haploid (n) sets of chromosomes. For example cells contain three (3n) or four (4n) haploid sets of chromosomes, which are called triploid and tetrapoloid cells, are polyploid. Polyploidy is very common in plant. It occurs naturally as a result of abnormalities of the cell cycle and/or meiosis. Polyploid cells and plants usually have larger size than the diploid counterparts. For this reason, creation of polyploid varieties has been considered an important strategy to improve the economic value of agricultural plants. Treating mitotic-dividing cells with colchicine has been used routinely in plant breeding lab to induce polyploidy in plant. Colchicine (C22H25NO6) is an alkaloid, originally extracted from the corm and other plant parts of the autum crocus, Colchicum autumnale. This drug disrupts the microtubles, preventing the formation of the spindle. As a result, the duplicated chromosomes fail to separate during mitosis. Mitosis that takes place during the treament with colchicine is called a “C-mitosis”. The success of colchicine treatment depends on the plant species, type of organs or tissues used.

II. Objective Upon completion of this investergation, the student should be able to: • Describe and explain how to induce polyploidy in plant by colchicines, and • Compare and contrast the behavior of chromosomes during C-mitosis and normal mitosis.

III. Material Onion bulbs

Water

0.05 % colchicine

Sand

Carnoy’s solution

Tissue papers

Arcetocarmin stain

Watch glasses

1N HCl

Microscope

90% & 70% ethanol

Microscope slides and coverslips

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IV. Procedure 1.

Before the practical, colchicine treatment of onion roots was performed with the following procedure: • Grow onions in the clean sand until the roots reach about 1 cm in length • Wash the roots with water and immerse them into 0,05 % colchicine for 2 hours. • Use scissors to collects the root tips. • Soak the roots tips in carnoy’s solution for 12 hours. • Wash in 90% ethanol twice for 10 minutes each time. • Store in 70 % ethanol until use.

2.

Each student shall prepare one specimen of colchicines-treated onion root tip using the same procedure as for preparation of specimen of the normal onion root tips in practical 2.

3.

Observe the specimen under low power and then under high power.

V. Report 1.

Identify and draw cells at various stages of C-mitosis (remember to label your drawing clearly).

2.

Do chromosomes in the C-mitotic metaphase align on the metaphase plate as those of the normal mitotic cells? Why?

3.

How can you differentiate between cells at C-mitotic anaphase with those at C-mitotic metaphase?

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LAB 4: MEIOSIS I. Introduction Gametes, cells specialized for sexual reproduction, are haploid, carrying only a single set of chromosomes and thus, only a single copy of the organism genetic information. Gametes are formed by the process of cell division called “meiosis”. This division process results in the reduction of chromosome number from diploid in the mother cells to haploid in the daughter cells. This reduction in chromosome number is essential because when two gametes fuse in fertilization, the chromosome number is restored to diploid in the embryo. Like mitotic process, meiosis is preceded by an interphase, when genomic DNA of the cell is replicate, doubling the number of chromosomes. The interphase is followed by two rounds of cell divisions, namely meiosis I and meiosis II. Each of these divisions is divided into prophase, metaphase, anaphase, and telophase, which can be differentiated by the roman number following the name of each phase (Fig. 4.1). The period between meiosis I and meiosis II is “interkinesis”, in which the nuclear membrane re-forms around the chromosomes clustered at each pole, the spindle breaks down, and the chromosomes relax. The behavior of chromosome during meiosis (Fig. 4.1) is summarized as follows: Prophase I; Prophase I is a lengthy stage, in which chromosomes contract and become visible; homologous chromosomes begin to pair up forming tetrads or bivalents; and crossing over (synapsis) take places. Near the end of prophase I, the nuclear membrane breaks down and the spindle forms. Metaphase I: The tetrads move toward the center and line up on the metaphase plate. As tetrads align themselves in the middle of the cell, they attach to spindle fibers in a unique manner: the centromeres of homologous chromosomes attach to separate spindles, each from different poles. Nuclear envelope completely disappears. Anaphase I: The unique even occurring at this phase is the separation of the homologs. In contrast to mitotic anaphase, the centromeres of a given chromosome do not divide. As a consequence, chromosomes of each homologous pair move toward opposite poles, resulting in the halving of chromosome number in the daughter cells.

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Telophase I: The chromosomes arrive at the spindle poles and cytoplasm divides. The chromosomes, however, do not completely uncoil. Prophase II: The chromosomes recondense; the spindle re-forms; and nuclear envelope once again breaks down. Metaphase II: Individual chromosomes line up on the metaphase plate with sister chromatids facing the opposite poles. Anaphase II: The kinetocores of sister chromatids separate and the chromatids are pulled to the opposite poles. Each chromatid is now a distinct chromosome. Telophase II: The chromosomes arrive at the spindle poles; nuclear envelope reforms around the chromosomes; and cytoplasm divides. The chromosomes relax and are no longer visible.

Figure 4.1 Meiosis process

II. Objective Upon completion of this investigation, the student should be able to • Describe the main events of meiosis, • Identify and draw cells in garlic chive flower at different meiotic phases, and 17

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• Compare and contrast between meiosis and mitosis.

III. Material Garlic chive flower

Microscope

70 % Ethanol & 90% Ethanol

Microscope slide & coverslip

Carnoy’s solution

Dissecting needle

Acetocarmine stain

Forceps

1N HCl

Immersion oil

10% glycerol

IV. Procedure Before the practical, the instructor collected garlic chive flowers and soaked them in carnoy’s solution for 12-20 hours. The flowers were then washed twice with 90% ethanol (10 minutes each time) and stored in 70% ethanol. 1.

Each students use a forceps to transfer some garlic chive flowers into a watch glass, and use dissecting needles to remove the perianth and petals from the flowers, only retain the anthers.

2.

Wash the anthers with water 3 times.

3.

Completely remove water from the watch glass. Be careful, not pour the tiny anthers down the sink.

4.

Add several drop of 1N HCl solution into the watch glass and soak the anthers in this solution for 10 minutes.

5.

Wash the anthers with water 3 times.

6.

Completely remove water, then add several drops of acetocarmine stain into the watch glass.

7.

Soak the anthers in staining solution for 30 minutes.

8.

Transfer four stained anthers to a microscope slide, which is pre-mounted with one drop of 10% glycerol. Cover with a coverslip.

9.

Use the handle of dissecting needle to gently apply pressure over the coverslip. This should squash the anthers into a thin cell layer.

10. Observe the specimen under low power objective to identify the area with many dividing cells. Then use higher power objective lens to carefully observe cells with different meiotic phases.

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V. Report 1.

Draw and clearly label cells at various stages of meiosis that you can observe.

2.

Compare the similarities and differences between the mitosis and the meiosis.

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LAB 5: POLYTENE CHROMOSOME OF DROSOPHILA MELANOGASTER I. Introduction Polytene chromosomes are extremely large chromosomes that are found in the insects of the order Diptera. These chromosomes are formed when cells undergo endomitosis, in which DNA repeatedly replicate without separation into the daughter cells. For unknown reasons, the centromeric regions of the chromosomes do not separate but bundle together in a mass called the chromocenter. Polytene chromosomes are usually found in the larval stages, where it is believed that polyploidy allows much faster larval growth than if the cells remained diploid. This is simply because each gene exists at high copy number in the polytene chromosome, resulting in higher rate of transcription. After stained, polytene chromosomes appear into banded-structure. Scientists correlate the size, width, and location of the bands with specific genes. Like the cells of the adult fruit flies, the cells from larval salivary glands have four pairs of homologous chromosomes: one pair of sex chromosome and three pairs of autosomes, which are numbered 2 to 4 (Fig. 6.1).

Figure 6.1 The polytene chromosome in Drosophila salivary gland.

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II. Objective Upon completion of this investigation, the student should be able to • Prepare the aceto-orcein specimen of Drosophila salivary glands, and • Recognize

Drosophila

polytene

chromosomes

and

discuss

their

significance.

III. Material Fruit fly larvae Aceto-orcein stain Water Paper towels Petri dish Microscope Microscope slides & cover slips Teasing needle

IV. Procedure A. Dissecting the salivary glands from the larva 1.

Use a teasing needle to transfer a big larva from the stock bottle into a drop of water on a clean microscope slide.

2.

Place the slide on the stage of a stereomicroscope.

3.

Dissect the larva under appropriate magnification. Place one teasing needle in the middle of the larva and the other needle at the anterior end, near the black mouthparts.

4.

While holding the larva with the first needle, pull outward with the needle at the anterior end of the larva. This will cause the internal organs of the larva to be pulled out of the body wall. The two transparent salivary glands located anteriorly, and usually bound to the edge by narrow, opaque ribbons of fat (Fig. 6.2).

5.

Use teasing needles to remove all part of larva including the fat from the salivary glands. Take care to avoid the water evaporates completely from the slide at any time during the dissection. 21

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Figure 6.2 Dissection of the salivary glands from Drosophila larva. B. Staining 1.

Use tissue papers to absorb water on the slide. Take care to retain the salivary glands on the slide.

2.

Quickly put a drop of aceto-orcein stain on top of the salivary glands for 30 minutes. Cover the slide with the petri dish to prevent the evaporation of the stain.

3.

Place a cover class over the drop of stain, gently press the handle of the teasing needle on the cover class to squash the salivary glands, rupture the nuclear membranes, and free the chromosomes.

4.

Observe the slide under the microscope at low- and high power.

V. Report 1.

Draw the observed polytene chromosome.

2.

Can you see the banding pattern of the chromosome? .............................................

3.

Can you locate the chromocenter? ............................................................................ 22

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4.

Discuss why the giant chromosomes have different structure from the normal chromosomes

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LAB 6: EXTRACTION OF DNA FROM PLANT CELLS I. Introduction DNA is genetic material of the cells and organisms. All genetic information of cells or organisms is included in DNA. DNA extraction from plant tissue can vary depending on the material used. Essentially any mechanical means of breaking down the cell wall and membranes to allow access to nuclear material, without its degradation is required. For this, usually an initial grinding stage with liquid nitrogen is employed to break down cell wall material and allow access to DNA while harmful cellular enzymes and chemicals remain inactivated. Once the tissue has been sufficiently ground, it can then be resuspended in a suitable buffer, such as SDS. In order to purify DNA, cell debris then will be precipitated by centrifugation. DNA must then be precipitated from the aqueous phase and washed thoroughly to remove contaminating salts. The purified DNA is then resuspended and stored in TE buffer or sterile distilled water. This method has been shown to give intact genomic DNA from plant tissue. To check the quality of the extracted DNA, a sample is stained with ethidium bromide, and visualised under UV light.

II. Objectives Upon completion of this investigation, the student should be able to -

Extract DNA from plant cells

-

Recognize the presence of DNA in the extraction.

III. Tools and Materials: 200mg plant tissue Eppendorf Mortar and Pestle Microcentrifuge Absolute Ethanol (ice cold) 70 % Ethanol (ice cold) 24

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7.5 M Ammonium Acetate 550 C water bath Water (sterile) Ethidium Bromide solution SDS buffer

IV. Procedure 1. Grind 200 mg of plant tissue to a fine paste in approximately 500µl of SDS buffer. 2. Transfer SDS/plant extract mixture to an eppendorf. 3. Incubate the SDS/plant extract mixture for about 15 min at 550C in a recirculating water bath. 4. After incubation, spin the SDS/plant extract mixture at 12000 g for 5 min to spin down cell debris. Transfer the supernatant to a clean eppendorf. 5. Add 250µl of Chloroform: Iso Amyl Alcohol (24:1) and mix the solution by inversion. 6. After mixing, spin the tubes at 13000 rpm for 1 min. 7. Transfer the upper aqueous phase only (contains the DNA) to clean eppendorfs. 8. To each eppendorf add 50µl of 7.5 M Ammonium Acetate. Mix by invert the eppendorf. 9. Add 500µl of ice cold absolute ethanol. 10. Invert the tubes slowly several times to precipitate the DNA. Generally the DNA can be seen to precipitate out of solution. 11. The tubes can be placed for 30 minutes to 1 hr at -200C after the addition of ethanol to precipitate the DNA. 12. Spinning the tube at 13000 rpm for a minute to form a pellet. 13. Remove the supernatant and wash the DNA pellet by adding 500µl ice cold 70 % ethanol and Spinning the tube at 13000 rpm for a minute. 14. Remove the supernatant. Wash the precipitate again with ice cold 70% ethanol. 25

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15. After the wash, Remove all the supernatant and allow the DNA pellet to dry (approximately 15 min). Do not allow the DNA to over dry or it will be hard to re-dissolve. 16. Resuspend the DNA in sterile DNase free water (approximately 50400µl H2O; the amount of water needed to dissolve the DNA can vary, depending on how much is isolated). - RNaseA (10 µg/ml) can be added to the water prior to dissolving the DNA to remove any RNA in the preparation (10 µl RNaseA in 10ml H2O). - After resuspension, the DNA is incubated at 650C for 20 min to destroy any Dnases that may be present and store at 40C. 17. Check the presence of DNA in the solution using UV light and Ethidium bromide.

V. Report. 1. Explain the function of SDS buffer? 2. Function of Amonium acetate? 3. What is the color of DNA after extration? 4. How do you know you extraction contain DNA?

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APPENDIX – RECIPES

Reagent composition Carnoy’s solution: -

Mix absolute ethanol and glacial acetic acid with the following ratio: 3 vol. of ethanol: 1 vol. of acetic acid

-

Prepare fresh.

Acetocarmin Carmin

0.1g

45% Glacial acetic acid

10ml

10% Ferric chloride (FeCl2*6H2O)

0.5ml

-

Boil 10 ml of 45% glacial acetic acid

-

Add 0.1 g of carmin.

-

Add 0.5 ml of 10% Ferric chloride (this step is optional).

-

Cool rapidly.

-

Filter and store in the dark.

Aceto-orcein Orcein

0.1g

Glacial acetic acid

5.5ml

ddH2O

~ 4.5ml

-

Boil 5.5ml of glacial acetic acid then add 0.1g of orcein powder.

-

Cool the solution.

-

Add distilled H2O to 10 ml and filter.

-

Store in the dark.

SDS buffer - Tris-HCl 1M, pH7.4

10ml

- EDTA 0.5M, pH8

2.5ml

- NaCl 5M

2.5ml

-SDS 10%

2.5ml

- ddH2O

32.5ml

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School of Biotechnology - IU

References 1. Genetics Laboratory Manual. Second edition. 2000. University of South Florida, Tampa. James R. Garey, Samantha R. Brown, Laurie L. Markham, Richard A. Anthony 2. TCU genetics lab manual. First edition. 2003. Texas Christian University. Phil Hartman and Misti Caudle. 3. Laboratory manual for bios 308. Genetics. Dept. of Biological Sciences. Northern Illinois University. DeKalb IL 60115. USA. Mitrick A. Johns.

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