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Experiment 6: Enzyme Activity
Summarize the characteristics of enzymes.
Show how changes in pH and temperature affect the rate of enzyme activity.
Enzymes are proteins that function as biological catalysts. They bring about all the activity within living cells. Without them, the metabolic reactions we depend upon for life would not proceed at all, or would occur so slowly that our cells would be unable to function. Changes in temperature and pH can easily disrupt both the hydrogen bonds and hydrophilic-hydrophobic interactions which are necessary in maintaining the three-dimensional configuration associated with functional enzymes. The destruction of the shape of an enzyme results in making that enzyme incapable of carrying out its normal catalytic capabilities.
Section B – Recycling of Enzymes
Dropper test tube (1)
Glass marking pencil hydrogen peroxide
Metric ruler small pieces of potato
Obtain one test tube and mark off from the bottom of the test tube, the heights 4 and 8 cm.
Add hydrogen peroxide with the dropper up to the 4 cm mark. Then add 2-3 small pieces of potato (about the size of an eraser).
Record your observations.
As the reaction appears to slow down, add additional hydrogen peroxide up to the 8 cm mark. Do not add any more potato.
Pour out all hydrogen peroxide. Cover potatoes with h2O. Boil water/potato mix by passing tube through flame of burner. Once boiled pour off water keeping potatoes in tube. Add more hydrogen peroxide to tube.
How did you know when hydrogen peroxide was being decomposed?
Release of bubbles indicated oxygen gas was being generated which is a product of the decomposition of Hydrogen peroxide
Catalase is found in potato cells. Why did the reaction continue after more hydrogen peroxide was added, even though additional potato chunks had not been added?
Because the catalase was present and is reusuable
What happened to reaction once potatoes were boiled? Why?
Boiling changes bonds in molecules and caused the catalase to be denatured. If an enzyme shape is changed then the active site also has changed shape and can no longer bind to the substrate(hydrogen peroxide)
Experiment 7: Photosynthesis
Understand the equation for photosynthesis.
Know how to separate the photosynthetic pigments and test the production of starch.
Photosynthesis occurs in the chloroplast of the leaf. Pigments located inside chloroplasts must absorb light. In this procedure, you will use a technique known as paper chromatography to separate and identify four major plastid pigments of green plants. In chromatography, a mixture of substances is concentrated in a line on very clean, porous paper. Then the paper is placed with one edge in a solvent so that solvent moves up the paper, like a wick, past the concentrated spot. Depending on its molecular structure and electrical polarity, a molecule will spend a certain percentage of its time dissolved in the readily moving solvent, and a certain percentage of its time absorbed to the fibers of the paper. So, if we select a solvent in which the solubilities of substances differ, they will separate from one another. A pigment’s molecular size, polarity, and solubility determine the strength of this tendency; pigments absorbed strongly move slowly, whereas those absorbed weakly move faster. The greater the distance the solvent travels, the clearer the separation between the different compounds.
The green plant pigments known as chlorophylls are never found alone. With them in the chloroplast are the fat-soluble carotenoid pigments which come in a variety of shades from yellow through orange to red. These are the pigments so conspicuous in tomatoes, carrots, squash and many other fruits. The major groups of carotenoid pigments are the carotenes and xanthophylls. In the procedure you will see four bands in the paper – a yellow band of carotenes, a yellow-orange band of xanthophylls, a blue-green band of chlorophyll a, and a yellow-green band of chlorophyll b.
The relationship of the distance moved by a pigment to the distance moved by the solvent front is specific for a given set of conditions. We call this relationship the Rf and define it as follows:
Distance moved by pigment
Rf = _____________________________________
Distance from pigment origin to solvent front
Thus, paper chromatography can be used to identify each pigment by its characteristic Rf.
Photosynthesis is the light-dependent and chlorophyll-dependent conversion of carbon dioxide and water to sugar, water, and oxygen. Oxygen is released to the environment, and sugar is used to fuel growth or is stored as starch. Here is the summary equation for photosynthesis.
6CO2 + 12 H2O light C6H12O6 + 6H2O + 6 O2
Section A: Separating Plant Pigments
Spinach Mortar Filter paper Test tube Pipette
Chromatography solvent (9 parts petroleum ether; 1 part acetone)
Make a highly concentrated mixture of chlorophyll by grinding approximately 5 grams of plant matter with about 10 ml of acetone in a mortar.
Cut a 1 cm wide by 12 cm long strip of filter paper. Fold strip lengthwise so it will be able to stand by itself.
Using a pipette, add several drops of the concentrated chlorophyll mixture to the filter paper approximately 2 cm from the bottom of the filter paper strip. Confine the spot of mixture to as small an area as possible. But make it concentrated by allowing each drop to dry before adding the next.
Fill the bottle with 2 ml of chromatography solvent.
After the filter paper strips have dried, place them in the bottle (the plant extract spot should NOT be touching the solution). Place an aluminum foil cover on the test tube and observe the separation of the plant pigments. After the solvent has reached the top of the paper strip, remove the strips from the jar and let it dry.
Ideally, four horizontal bands should be present. Carotenes move the fastest, followed by xanthophylls, then chlorophyll a, and last, chlorophyll b. Label each band you observe.
Calculate the Rf values for each pigment and record below. (show calculations please)
Results from procedure above Results from pigment transfer using coin
What does a small Rf value tell you about the characteristics of the moving molecules?
That the pigment was not strongly attracted to solvent compound and did not move very far
Is it possible to have an Rf value greater than 1? Why or why not?
No, because having an rf greater than 1 would indicate that the pigment molecule moved farther than the solvent which is impossible because the solvent caused the pigment to move
What four pigments are found in most plants?
Which pigment seems to be the most abundant? The least?
Which procedure used yielded the best results? Why do you think this happened?
Experiment 8: Mitosis & Meiosis
Understand the relationship between chromosomes and DNA molecules.
Describe events associated with the cell cycle.
Identify the five phases of the cell cycle; describe the events that highlight each stage.
Describe the events of meiosis.
Compare and contrast meiosis and mitosis.
Understand the most significant events of meiosis.
The process of growth and division in a typical eukaryotic cell is called the cell cycle and is composed of five phases. The G1 phase is the cell’s primary growth phase while the genome is replicated during the S phase. During the G2 phase various organelles are replicated. M (mitosis) phase is nuclear replication in eukaryotic cells. It lasts for less than 10% of the time of the cell cycle. C (cytokinesis) phase is the division of the cell and cytoplasm into halves, each half containing a nucleus.
The stages of mitosis are prophase, metaphase, anaphase and telophase. During early prophase, the chromosomes condense, centrioles begin to migrate to opposite sides of the nucleus and the nuclear membrane begins to disintegrate. In mid-prophase spindle fibers, originating from the centrioles, attach to the centromere of the duplicated chromosomes. As the cell progresses toward late prophase, the chromosomes are manipulated toward the center (mid-plant) of the cell. Once the chromosomes are lined up along the mid-plant of the cell, the cell is in metaphase. In anaphase, the centromere of the duplicated chromosomes divide and the spindles pull the chromosomes to opposite sides of the cell. The cell now enters telophase. During telophase, the nucleus reforms, the chromosomes unwind and the centrioles divide. The cell may now undergo cytokinesis and physically split to form two cells. It is possible for a cell to undergo mitosis without going through the process of cytokinesis, but these cells will possess two nuclei.
Chromosomes are of Deoxyribonucleic acid (DNA) which contains the genetic information. DNA is in the shape of a double helix. The sides of the helix are composed of alternating molecules of deoxyribose sugar and phosphate molecules that are held together by strong covalent chemical bonds. Attached to each side chain is a nitrogen base. Nitrogen bases are either adenine and guanine, or the thymine and cytosine. Nucleic acids along the length of the DNA molecule are arranged in pairs which are held together by weak hydrogen bonds. Adenine is joined to thymine by two hydrogen bonds and guanine to cytosine by three hydrogen bonds. A sequence of nucleic acids along the length of the DNA that carries the genetic information for the production of a particular product is called a gene. Genes dictate all the traits that each organism expresses.
onion root tip slides whitefish blastula slides
Model of mitosis
Cell division: mitosis
Obtain an onion root tip and a whitefish blastula slide.
Examine the cells first on low then high magnification. Note that some of the cells contain condensed and stained chromosomes. Sketch each phase below in the appropriate column animal vs. plant cells mitosis.
Examine the available model demonstrating mitosis.
MITOSIS *in box sketch what is happening in cell and to the right give a short description of each phase
Experiment 9: Human Genetics
In this lab we will identify some of the genetic characteristics you have. We will use the phenotype (the character seen on an individual) to predict your possible genetic make-up (genotype). You will determine the likely genotypes and phenotypes of offspring from reproduction (theoretical) with other class members, by using punnett squares. Remember, dominant genes are indicated by a capital letter and recessive genes are indicated by a lower case letter. Dominant genes are expressed (seen in the phenotype) in either the heterozygous or homozygous condition. Recessive genes are only expressed in the homozygous condition.
Some Important Definitions for this Lab
Gene………………………. a specific segment of DNA that codes for a particular trait
Allele……………………… one, two, or more forms of a gene for a particular trait found at a given location on a chromosome
Dominant Gene……… masks the effect of a recessive gene, only one needs to be present for the trait to be expressed
Recessive Gene……… is not expressed when the dominant allele is present, a recessive trait is only expressed when both alleles present are recessive
Homozygous…………. identical alleles for a particular trait, two dominant or two recessive alleles for a particular trait
Heterozygous……….. having two different alleles present for a particular trait, such as, one dominant and one recessive
Genotype……………… the types of genes present in an individual
Phenotype……………. the characteristics present on an individual
Determine your phenotype and possible genotype for the following characters:
Bent little finger
Hair on back of hand
Can taste (Pt)
No hair (m)
Cannot taste (pt)
Ear lobes are attached either along their entire length to the side of the head or they dangle free (Pendulous). The “free” condition is dominant (P) over the attached (p). Ask a neighbor to tell you which condition you have and record your results on Table 2.
Curved Little Finger
The last joint on the little finger may be straight or bent toward the ring finger. The bent condition is dominant (C) over the straight condition (c). Compare your little finger with those of others and record your phenotype for this trait on Table 2.
Hair on the Back of the Hand
Hair on the back of the hand (M) is dominant over no hair on the back of the hand (m). Record your condition on Table 2.
The ability to roll the sides of the tongue upward to form a rough tube shape is dominant (T) over the inability to perform this action (t). Record your condition on the table.
The presence of freckles (F) is dominant over the absence of freckles (f). However, the expression of freckles can be influenced by complexion, skin pigmentation, or the amount of time your skin is exposed to the sun. Record your results.
Loose ligaments which permit the thumbs to be thrown out of joint are dominant (L) over tight ligaments (l). If you have loose ligaments in your thumb joints you will be able to bend the last thumb joint back at a 60◦ angle or more. See if anyone in the lab section has the condition, if you don’t. Record your trait.
Eye color is based on the amount of pigment present on the front part of the iris of the eye. Brown represents the dominant condition (B) in which pigment is present. Blue (b) represents the recessive condition where no pigment is present. There are gradations between these two colors because they are influenced by other genes. However, we will treat this trait as if it were entirely determined by a single pair of genes. To prevent confusion, all those with hazel, green and gray eyes should classify themselves as dominant. Record your results.
Look at the hairline on your forehead (use a mirror). If you have a triangular shaped downward dip in your hairline in the middle of your forehead, you have a Widow’s Peak. This condition is dominant (W) over the absence of a Widow’s Peak (w). Record your results.
Your laboratory instructor will give you a piece of paper which has been soaked in phenylthiocarbamide (PTC). Ability to taste PTC is dominant (Pt) for the recessive expression. Record your results.
Table 2: Fill in your phenotype and possible genotypes.
A. Ear Lobes
B. Bent Little Finger
C. Hair on Back of Hand
D. Tongue Rolling
F. Double-jointed Thumbs
G. Eye Color
H. Widow’s Peak
I. PTC Tasting
Now for each character A-I, pick a potential mate from the class and determine using punnett squares the likelihood of different genotypes and phenotypes.
Your phenotype ____________________________________________
Your possible genotypes _____________________________________
Possible genotypes ___________________________________
Possible genotypes _______ _______ _______ _______
Possible phenotypes _______ _______ _______ _______ _______ _______
Ratio of phenotypes _______ _______ _______ _______ _______
Bent Little Finger
Your phenotype ______________________________________________
Your possible genotypes _______________________________________
Possible genotypes _____________________________________
Your phenotype _________________________________________
Your possible genotypes __________________________________
Possible genotypes _____________________________
Your phenotype ________________________________________
Your phenotype ______________________________
Red-green color blindness. Color blindness is sex-linked, and therefore genotypes are dependent upon sex, as shown in the table below.
Table 13.3. Color Blindness Phenotypes and Genotypes
A woman with normal vision marries a man with normal vision and their first child is color blind.
Womans genotype xCxC
Mans genotype xCY
I. What is the genotype of this mother?
II. If their next child is a girl, what is the likelihood she will be color blind?
0% neither girl is going to be colorblind. One is a carrier and the other is normal
III. What is the likelihood that their next son will be color blind?
The woman’s husband dies and she remarries a color blind man.
I. What is the likelihood their sons will be color blind?
50% one of two sons will be colorblind
II. What is the likelihood their daughters will be colorblind?
50% one of two daughters is colorblind
Experiment 10: DNA Structure and Isolation
What is DNA?
Obtain a DNA model kit from the supply table.
Working in groups of two, assemble a strand of DNA that is 4 deoxyribonucleotides long.
Draw your structure in the below and have your instructor check your strand and initial the star.
DNA Molecule Drawing. Label all parts.
DNA Isolation from plant cell.
Materials: Spinach( or some plant Water bath (hot and cold)
Dish detergent Distilled water
Salt Protease enzyme
(made with 5g of meat tenderizer and 95mL of h20)
Mortar and pestle Ethanol (freeze 24 hours before)
Test tube Pipettes, 10ml and 1ml
Beakers (2) Cheesecloth
Funnel Stirring rod
In a beaker put 3 grams of salt and 2 tsp. of dish detergent. Add 100ml of distilled water. Stir well to dissolve salt.
Put 2 tsp. of spinach in mortar and grind using the pestle. Grind until smooth.
Add the ground spinach to the salty dish detergent solution.
Place the beaker in a hot water bath at 60◦C for 15 minutes.
Cool the mixture by placing the beaker in an ice water bath for 5 minutes, stirring frequently.
Filter the mixture into a second beaker. Cheesecloth and funnel needed.
Dispense 10ml of the spinach extract in a test tube. Add 3 drops of protease enzyme. Mix well.
Carefully pour 10ml of ice cold ethanol down the side of the test tube to form a layer on top of the spinach extract. Place the tube in a test tube rack for a few minutes without disturbing it.
DNA will form in the upper (ethanol) layer.
Scoop out the DNA as it forms.
DNA Isolation Questions
Instructions: Answer the following four questions on the spaces below. You may use your notes or the book for assistance. Each class member will hand in their own work.
Why is DNA extraction done?
See this animation:
to determine paternity, analyze crime scene evidence, inheritance of conditions
Describe the major differences between the plant and animal cell.
Animal cell: no cell wall, has centrioles,
Plant cell: has cell wall, chloroplasts, central vacuole, no centrioles
Why was it necessary to place the spinach into the blender?
Break open cell walls
Why do we treat the cells with soap?
Break open cell membranes
Why do we add salt?
What is a protease enzyme? Why was the spinach extract treated with a protease?
A protease is a compound that degrades proteins. It helped to break dna away from histone proteins
How does the DNA of a eukaryote differ from a prokaryote?
Dna in euk. Is wrapped around histone, dna is linear and exists in much larger quantities.
Prokaryotes dna is not inside a membrane has only a circular chromosome.olor blind?