TB20003 GENETICS Lab 1: Mitosis in Onion Root Tips

Introduction All new cells come from previously existing cells. New cells are formed by the process of cell division which involves both replication of the cells nucleus (karyokinesis) and division of the cytoplasm (cytokinesis) to form two genetically identical daughter cells. There are two types of nuclear division: mitosis and meiosis. Mitosis typically results in new somatic (body) cells. Formation of an adult organism from a fertilized egg, asexual reproduction, regeneration, and maintenance or repair of body parts is accomplished through mitotic cell division. Meiosis, on the other hand, results in the formation of either gametes (in animals) or spores (in plants). These cells have half the chromosome number of the parent cell. Where does one find cells in the process of mitosis? Plants and animals differ in this respect. In higher plants the process of forming new cells is restricted to special growth regions called meristems. These regions usually occur at the tips of stems or roots. In animals, cell division occurs almost anywhere as new cells are formed or as new cells replace old ones. In both plants and animals, though, tissues rarely divide once the organism is mature. Phases of cell division: 1) Interphase is considered the first and last stage of plant cell division. It is the stage in which the cell is growing in size and replicating its DNA in preparation for division. The nucleus is apparent. 2) Prophase. During Prophase the nuclear envelope starts to break down and all the chromosomes start to coil up in the center of the cell. 3) Metaphase is the middle stage at which point all the chromosome pairs line up in the center of the cell along spindle fibers that pull to either side of the cell. 4) Anaphase. The spindle fibers become shorter and pull each chromosome pair apart to the opposite ends of the cell. 5) Telophase. The final stage of cell replication.The nuclear envelope is reformed. Cytokinesis takes place. A new cell wall is created down the center and two daughter cells are formed. Objectives Stain tissue for the identification of cells in the various stages of mitosis Study the structure of chromosomes Study the movement of chromosomes during mitosis Identify the stages of mitosis in plant cells

Materials Allium cepa (onion root tip) Glass slides Cover slips Scalpel Glass rod Blotter Tissue Forceps Petri plate Distilled water 1M HCl Aceto-carmine stain Compound microscopes Procedure 1. Onion bulbs have been rooted in water. Growth of new roots is due to the production and elongation of new cells. Mitotic divisions are usually confined to the cells near the tip of the root. 2. Obtain an onion bulb that has been rooted in water. Cut 2 or 3 roots off near the base of the bulb. 3. Then, cut off the bottom 1 or 2 mm of the root tip and place it in a petri plate. With a pasteur pipette, add a small puddle of 1M HCl and let root tip stand for 4 min. 4. Rinse with water and blot until the water is clear. Remove the root tip from the HCl with forceps and place on a slide. 5. Cover the root tip with 2 drops of aceto-carmine stain and let sit for 2 minutes. 6. Squash the root tip, on each slide, pressing straight down so as not to overlap the cells by using the glass rod. 7. Place two more drops of stain upon the root tip and wait for another two minutes. 8. Then place cover slip flat upon the root tip, making certain not to move the cover slip horizontally. 9. Press the cover slip gently with a pencil eraser, again only straight down without moving the cover slip. 10. Mount the slide on your microscope.

11. Use the low power objective on your microscope to look for thin layers of cells and then use the 40X power objective to observe mitotic stages in individual cells. 12. Identify chromosomes at the various stages of mitosis. 13. Make sketches of the mitotic stages observed. Questions: 1. Draw, label and explain the characteristics of each mitosis phase observed in this experiment. 2. Which stage(s) have the easiest chromosomes to see? Why is this so? 3. Why are the cells in mitosis located near the tip of the root? 4. What is the importance of mitosis towards plant growth? 5. What is the difference in the process of mitosis between animal and plant cells? 6. Explain the definition of chromatins, chromatids, chromosomes and centromers?

TB20003 GENETICS Lab 2: Meiosis

Introduction Meiosis is the second important kind of nuclear division. It resembles mitosis in many ways but the consequences of meiotic divisions are very different from those of mitotic divisions. While mitotic division may occur in almost any living cell of an organism, meiosis occurs only in special cells. In animals, meiosis is restricted to cells that form gametes (eggs and sperm). Each species has a characteristic number of chromosomes per somatic cell. Fruit flies have 8; normal humans have 46. They exist as homologous pairs (partners) that are similar in size and shape and carry the same kinds of genes. Thus humans have 23 homologous pairs. The full complement of 46 chromosomes is referred to as the diploid number (referring to the fact that each kind of chromosome is represented twice). In higher organisms when an egg is fertilized, the egg and sperm fuse to form a single cell called a zygote which develops into a new organism. If the egg and sperm were both diploid (46 chromosomes each in the case of humans) then the resulting zygote would be tetraploid. This would be an intolerable situation, so a mechanism has evolved to insure that each gamete (egg or sperm) contains only one representative of each homologous pair (or half the diploid number). This is referred to as the haploid number. Haploid Egg + Haploid Sperm = Diploid Zygote The mechanism that makes this possible is meiosis. Meiosis consists of two divisions, Meiosis I and Meiosis II, and can potentially result in the production of four cells. However, the DNA is only synthesized once (prior to Meiosis I). The subdivisions of meiosis are named like the subdivisions of mitosis (prophase, metaphase, anaphase, telophase) but the events are somewhat different. Objectives To study the events associated with meiosis. Materials Slides of rice paddy cells with different meiosis stages Procedure 14. Mount the slides on your microscope. 15. Use the low power objective on your microscope to look for thin layers of cells and then use the 40X power objective to observe meiosis stages in individual cells. 16. Identify chromosomes at the various stages of meiosis. 17. Make sketches of the meiosis stages observed.

Questions: 7. Draw, label and explain the characteristics of each meiosis stage observed in this experiment. 8. How does the outcome of meiosis differ from that of mitosis? 9. What is recombination, and in what stage of meiosis does it occur? 10. Which of the two meiotic divisions (meiosis I and meiosis II) is similar to a mitotic division? 11. Provide two reasons why meiosis leads to genetic variation in diploid organisms.

TB20003 GENETICS Lab 3: Mendelian Genetic Corn Kernel Analysis

Introduction Mendelian genetics is built on the work done by the "father of genetics", Gregor Johann Mendel (1822-1884). His contributions to the study of inheritance paved the way for our basic understanding of how traits are inherited from one generation to the next. Mendel did much of his work with easily obtained local organisms, especially members of the genus Pisum or garden peas. Mendel recognized from observations and early experiments that his experimental organisms had two alternate forms of a trait. We call these alternate forms alleles (genes). According to Mendel, an individual possesses two alleles for each trait. The individual can have two alleles that are the same or one of each form. If the individual possessed two alleles that were the same the individual would be homozygous (or pure) for the trait. Mendel would indicate this by using letters to represent the alleles. If an individual was homozygous then it would be AA or aa. If the offspring contained one of each allele then it was termed heterozygous or Aa. The uppercase form of the allele was used to indicate that the individual was dominant for the trait and lower case indicated that the individual was the contrasting opposite or recessive for the trait. The terms dominant and recessive are used to signify when the trait appeared in the offspring of a cross between two homozygous individuals that represented alternate forms of the trait. If the trait appeared in the first generation (first filial or F1) then it was designated as dominant. If the trait "skipped" the F1 and appeared in the second generation (F2) then the trait was designated as recessive. The F2 generation was obtained by crossing two F1 individuals. The corncob is not the fruit of the corn plant in itself, nor is the kernels the seeds. Each kernel of corn is really a fruit, which develops from the ovary of one of the female flowers of the plant. There are a great number of inheritable characteristics in corn (Zea mays). In this experiment we will investigate two, the color of the kernel and starchy consistency of the endosperm which gives rise to wrinkled or smooth kernels. The endosperm is a nutritional reserve for the developing corn seedling that provides energy to the seedling after immediately germination. This reserve is drawn on until the developing plant begins to generate its own energy by photosynthesis. Three layers of cells protect the endosperm. The inner most layer, the aleurone layer, contains purple pigments called anthocyanins. The amount of anthocyanin in the aleurone layer and the amount of starch present in the endosperm are genetically determined and can be inherited according to Mendelian rules. Objectives 1. To determine the quantities of purple and white corn kernels correspond to the Mendelian ratios of the F2 generation of a monohybrid cross, and 2. To determine the quantities of yellow and white and wrinkled and smooth kernels correspond to the Mendelian ratios of the F2 generation of a dihybrid cross.

Procedure (a) Purple and yellow corn kernel - The Monohybrid Crosses 1. You will be provided with an ear of corn whose kernels show two different colors-yellow and purple. 2. Count and record the numbers of purple and yellow corn kernel. All individuals in your group should individually count the kernels. Record the results of the counts in table 1 below. Table 1 Kernel Phenotypes Total Kernels Group 1 Total Kernels Group 2 Total Kernels Group 3 Total Kernels Group 4 Total Counts 1 + 2 + 3 + 4

Purple

Yellow

3. When your groups have finished counting enter all of the groups totals in the table 1. When everyone has entered their data into the table, combine the totals for each phenotype counted and record them "Class Total" columns for table 2. Table 2 Kernel Phenotype Class Number of individuals (actual counts) Expected number of individuals

Purple

Yellow

Class Totals

4. Data Interpretation Examine the totals obtained by the class. Using the kernel counts, what is the dominant phenotype? How do you know? 5. The corn kernels are the F2 generation resulting from a cross between a homozygous purple corn (PP) and a corn that is homozygous recessive white (pp). Fill in the following Punnett square to show how the ears were produced. Parents PP x pp Purple white F1 ____/____ F2 ____/____ ____/____ ____/____ ____/____ 6. Determine the expected numbers for each phenotype using Mendels law of segregation and perform a Chi-square calculation to determine if the class data obeys Mendels law of segregation. Include a statement describing what the Chi-square calculation indicates about the class data.

(b) Purple-Yellow-Smooth-Wrinkled Corn Kernel Dihybrid Crosses 1. You will be provided with two ears of corn. One will be from the F2 generation which is dihybrid crosses of purple-smooth and yellow-wrinkled parental. One more corn which is the progeny of one of the F1 generation with the parental plant. (You will not be told which corn is from the F2 generation and which corn is the progeny from the experimental crosses). 2. Count and record each phenotype on both the corns. 3. Count the phenotypic ratio of both corns. 4. Determine which corn is from the F2 generation. 5. Determine the predicted phenotype of the F1 generation. 6. Explain the dihybrid crosses with figure. Indicate the genotypes of each generation: parental (P), F1 and F2. 7. Calculate the genotype frequency of F2 generation. 8. Determine the number and phenotypic ratio of the corn from the experimental crosses. Which parental plant was crossed with the F1 plant to get the mentioned corn? Explain the crosses with figure, include the genotypes. 9. What is the law of independent assortment? Explain the phenotypic ratio obtained from the F2 generation by using Mendel Law.

TB20003 GENETICS Lab 4: Mendelian Genetic Human single gene traits

Purpose To develop an understanding of basic Mendelian inheritance as it applies to human single gene traits. Introduction All people are recognizably human, but no one is exactly like anyone else, not even an identical twin. The basis for the similarity and the reasons for the diversity that coexist in all species have puzzled and intrigued people for thousands of years. Several human traits may be used to demonstrate the individuality in humans. They are controlled by a single gene with two alleles; each allele producing a distinct phenotype. Alleles are different expressions of the same gene. All can be used to demonstrate Mendel's Law of Segregation. Procedure: 1. In this activity you will be examining NINE easily observable traits. Assume that each trait is controlled by a single pair of alleles. For each trait described, record your phenotype and genotype in TABLE I. Use the letter symbols given for each genotype. Indicate a dominant phenotype by a single capital letter followed by a blank space (example: A_). Indicate a recessive phenotype by the use of two lower case letters (example: aa). Pictures of these traits can be found in FIGURE I. 2. Tongue rolling - The ability to roll the tongue is dominant (R), while non-rolling is recessive (r). It is debatable whether this is genetic. Record your phenotype and genotype in TABLE I. 3. Free ear lobe - In most people the ear lobes hang free. This is the dominant trait (E). The attached earlobe is recessive (e). Record your observations. 4. Hand clasping - Clasp your hands together. Notice whether your left or your right thumb is on top. If the left thumb is on top you have the dominant trait (C), the right thumb is recessive (c). Record your observations. 5. Hitchhiker's thumb - Hold out your hand and make a fist with the thumb extended. Bend the last joint of the thumb back as far as possible. A straight thumb is dominant (S) while a bent thumb is recessive (s). Record your observations. 6. Bent little finger - Place the palms of your hand gently together, side-by-side, with the palms facing upward. The dominant condition is for the last two joints of the little fingers to bend away from each other (B), while straight little fingers are recessive (b). Record your observations. 7. Widow's peak - The action of a dominant gene (W) results in a hairline that forms a distinct point in the middle of the forehead. The straight hairline is recessive (w). Record your observations.

8. Dimpled chin - A cleft in the chin is a dominant trait (D) while the absence of a cleft is recessive (d). Record your observations. 9. Mid-digital hair - Each of your fingers is composed of three segments. If any hair grows on the middle segments, you have the dominant allele for mid-digital hair (H). If you do not have any hair you are recessive (h). Record your observations. 10. Pigmented irises - When a person is homozygous for the recessive gene (p), there is no pigment in the front part of the eyes. and a blue layer at the back of the iris shows through, resulting in blue eyes. A dominant allele of this gene (P) causes pigment to be deposited in the front of the iris, thus masking the blue to various degrees. A dark iris pigment (green/brown/black) is dominant over the light pigmentation. (gray/blue). Record your observations.

Question: 1. Calculate the frequency of each phenotype of the human single gene traits. 2. Is it true that dominant phenotypes are always the most common in a population? Explain your answer. 3. Is it possible to determine the genotype of a person showing a dominant phenotype? A recessive phenotype? Why? 4. A confusion happened for determining parents for two babies in a hospital, therefore blood typing was conducted and the results were as follows: Baby 1 = O blood type Baby 2 = A blood type Ali = AB blood type Alis wife = B blood type Ahmad = B blood type Ahmads wife = B blood type With the help of appropriate figures, determine which babies belong to which set of parent (include genotypes of each person).

TB20003 GENETICS Lab 5: DNA Extraction from Onion

Introduction
DNA is present in the cells of living organisms. The first step in doing DNA analysis is isolating DNA from

cells. You will learn the basic concepts of DNA extraction that applies not only to onion but also to other samples. Please take note on the chemicals used and think about why they are used in particular steps. We hope you will enjoy yourself in this experiment.

Material: Per class 70% iced cold isopropanol Extraction solution (Lysis Solution) Ice chest containing ice Per group Onion 2 Falcon Tubes (15mL) (Label `P1 and `P2) 2 Conical Flasks (50mL) (Label S and SS) 500 ml beaker Glass Rod (2 pieces) Shampoo (4ml) Sterile Distilled Water (40ml) Table salt (0.3g) Hot Plate and Stirrer Procedure: (A) Extraction solution recipe:
Type of shampoo: `clear type, DO NOT use shampoos with conditioner or baby shampoo. Mix 4ml shampoo with 36ml distilled water. Stir well and slowly. Divide the mixture into two 50 ml conical flasks (20ml each) and label as `S and `SS. Add 0.3g salt into flask (SS). Dissolve the salt by stirring slowly to avoid foaming.

(B) Onion Extraction 1. Take an onion and cut into half. Slice into pieces and transfer approximately 2-3g into 2 tubes of 15mL Falcon tube and label the tubes as P1 and P2. 2. Add 6mL of extraction solution `S into Falcon tube `P1 and 6mL of extraction solution `SS into falcon tube `P2 by using plastic pipette. What do you think the shampoo solution does to the onion? 3. Blend/Homogenize the onions into small pieces by using homogenizer. What does blending/homogenizing the onion do? 4. Transfer 4mL of solution that contains DNA into new 15 mL falcon tubes by using plastic pipette. 5. Being careful add 4mL of ice-cold 70% isopropanol by using plastic pipette. What do you think the alcohol does? Why do we want it cold? 6. Being careful not to shake the tubes, put the plastic pipette inside the falcon tube and mix the solution carefully. 7. Take a look at your tube and observe what stick to the surface of the plastic pipette. What do you see on the surface of the plastic pipette? Question: 1. One way to purify a molecule is to get rid of everything but that molecule. If we want to isolate DNA from bacteria, what do we have to get rid of? 2. What materials would you use to do that? 3. What can we do with the DNA once we've purified it?

TB20003 GENETICS
Lab 6, 7 & 8: Mini Project (DNA of Fish) Introduction: This mini project consists of extraction, amplification and gel electrophoresis of plants. The objective of this mini-project is to provide an overall process of the DNA isolation. You should be able to correlate the concepts from previous laboratory session to the standard protocol provided here. Time: Lab 6: DNA Isolation (Day 1: 2 hours) Lab 6: Quantification (Day 1: hour) Lab 7: PCR (Day 2: Preparation: 1 hour; PCR Machine: 2 hours) Lab 8: Electrophoresis (Day 2 or 3: Preparation: 1 hour for gel to set; Gel electrophoresis: 1 hour)

(A) DNA Isolation Material:

2x CTAB buffer (50ml) [1.0g CTAB Powder (=2x); 5.0ml 1M Tris-HCl pH8 (=100mM) 2.0ml 0.5M EDTA(=20mM); 14.0ml 5M NaCl (=1.4M) P:C:IA (25:24:1 Phenol:Chloroform:Isoamyl alcohol) 20% SDS Isopropanol (-20C) Ice Cold 5M Potassium Acetate Ice Cold 70% EtOH Ice Cold 1xTE Buffer 1.5mL eppendorf tubes Scissor / Scalpel Set Thermomixer/waterbath (60 and 55C) Refrigerated Centrifuge

Procedure: 1. Using sterile scalpel minced fish muscle samples (50mg) and transfers into sterile 1.5mL eppendorf tube. 2. Add 600uL of 2x CTAB Buffer + 60uL of 20% SDS + 6uL of Proteinase K, mix by vortexing for 20seconds and incubate at 60C for1 hour. *Homogenize samples by using hand pastel for every 15 minutes incubation 3. Add 300uL of ice cold 5M Potassium Acetate, mix by vigorously vortexing for 30 seconds. 4. Centrifuge for 10 minutes, 13,000 rpm at 4C 5. Transfer 500uL supernatant to new sterile 1.5mL eppendorf tube containing 5uL of RNase A. 6. Mix samples by inverting the tubes for 10X and incubate at room temperature for 15 minutes. 7. Add 500uL of P:C:IA and mix samples by inverting 30X 8. Centrifuge for 5 minutes, 13,000 rpm at 4C 9. Transfer approximately 300uL of aqueous layer to sterile 1.5mL eppendorf tube. 10. Add 1mL of ice cold absolute isopropanol. Invert 30X to mix and incubate on ice for 10 minutes. 11. Centrifuge for 10 minutes, 13,000-15,000 rpm at 4C 12. Discard supernatant gently and add 1mL of ice cold 70% Ethanol 13. Centrifuge for 5 minutes, 13,000-15,000 rpm at 4C 14. Discard supernatant gently and air dry the tubes 15. Add 50-100l of TE buffer and put samples on ice for further use.

(B) Quantification To determine the DNA concentration using UV spectrophotometer. For double-stranded DNA: One OD260nm unit = 50 ng/l DNA

LAB 7: Polymerase Chain Reaction

1.

In a 1.5 ml tube on ice, prepare a mix for the number of PCR reactions that you want to perform plus one (i.e., if youre doing 10 reactions, make enough mix for 11) A standard recipe for one reaction is:

Component PCR Reaction Buffer MgCl2 dNTPs Taq Polymerase Forward Primer Reverse Primer DNA template Sterile dH2O

Stock Solution 5X 25mM 10mM 5U 100uM 100uM 50ng/ul -

Working Solution 1X 1.5mM 0.15mM 1U 10pmol 10pmol 50ng/ul TOTAL

1X 4.0 1.2 0.3 0.2 1.0 1.0 1.0 11.3 20uL

uL

2. 3.

4.

Dispense 19 l of reaction master mix into each sterile PCR tubes. Finally, to each tube, add 1l of the appropriate template DNA. Always remember to include a negative and a positive control Positive control: 19uL Mastermix + 1uL of DNA X (previously amplifying 16S rDNA) Negative control: 19uL Mastermix + 1uL sterile dH2O/TE buffer Put the tubes into the thermocycler and run the following profile: 16S rDNA Amplification:

Initial Denaturation Denaturation Annealing Extension Final Extension Forever *Set program as heated lid

96C 96C 50C 72C 72C 10C

1 min 30 sec 30 sec 1 min 1 min

35X

Lab 8: Agarose Gel Electrophoresis Materials 1. 2. 3. 4. 5. 6. Agarose Powder TBE/TAE buffer Agarose Gel Electrophoresis Tank, Trays and comb 1kb DNA ladder 6X Loading dye Micropipette White tips Parafilm Oven Staining solution Ethidium bromide (EtBr) Gel View Image Systems Alphaimager etc. Select a tray large enough for the number of samples that you are running. Tape off the sides of the tray if necessary and place the comb(s). Weigh the required amount of agarose (normally 1% gels are used, i.e. 1 g of agarose per 100 ml of gel buffer) and put it into an Erlenmeyer flask or a beaker. Add the required amount of gel buffer (TAE or TBE). Boil in the microwave oven: full power, 1 min per 50 ml, or on a hot plate; keep stirring to prevent the agarose from baking to the bottom of the flask. After boiling, add 1 l of ethidium bromide working solution (10 mg/ml) per 50 ml of gel. EtBr is a very powerful mutagen! Wear gloves at all times, and clean everything meticulously after use! Allow the gel to set and cool. Remove the tape and comb and put the gel in the electrophoresis tank. Make sure the electrophoresis buffer rises several mm above the surface of the gel. Cut a piece of parafilm and place it on a microfuge tube rack. Press small wells into the parafilm with the tip of a finger. Place 2l droplets of 6x loading buffer into each well (as many as you have PCR-reactions plus one [for the size marker]). Pipette 10 l of PCR-product into the loading buffer. Then pipette the entire amount into the slot in the gel. Be careful not to let the slot overflow. In the last slot, put a size marker (2 l is enough). Apply between 50 and 100 V across the gel. After a few minutes, make sure the samples are running in the right direction. When the samples have run far enough (when the blue colour is 3-6 cm away from the slots), switch off the power and take the gel out of the tank (gloves!). Put the gel on the UV tray and check for bands. If you want to use any PCR-products directly for cloning, put a thin layer of plexiglass under the gel to prevent DNA degradation. Photograph.

7. 8. 9.

10. 11. 12. 13. 14. 15.

Reference:
Murray, M.G. and Thompson, W.F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research. 8: 4321-4325.