# Biology labs

dmmontel
Module3Labs-Biology.doc

Pre-Lab Questions

1. What are chromosomes made of?

2. Research the differences that exist between mitosis and binary fission. Identify at least one difference, and explain why it is significant.

3. Cancer is a disease related to uncontrolled cell division. Investigate two known causes for these rapidly dividing cells and use this knowledge to invent a drug that would inhibit the growth of cancer cells.

Experiment 1: Observation of Mitosis in a Plant Cell

In this experiment, we will look at the different stage of mitosis in an onion cell. Remember that mitosis only occupies one to two hours while interphase can take anywhere from 18 - 24 hours. Using this information and the data from your experiment, you can estimate the percentage of cells in each stage of the cell cycle.

 Materials Onion (allium) Root Tip Digital Slide Images

Procedure:

Part 1: Calculating Time Spent in Each Cell Cycle Phase

1. The length of the cell cycle in the onion root tip is about 24 hours. Predict how many hours of the 24 hour cell cycle you think each step takes. Record your predictions, along with supporting evidence, in Table 1.

2. Examine the onion root tip slide images on the following pages. There are four images, each displaying a different field of view. Pick one of the images, and count the number of cells in each stage. Then count the total number of cells in the image. Record the image you selected and your counts in Table 2.

3. Calculate the time spent by a cell in each stage based on the 24 hour cycle:

 Hours of Stage = 24 x Number of Cells in Stage Total Number of Cells Counted

Part 2: Identifying Stages of the Cell Cycle

1. Observe the images of the root cap tip.

2. Locate a good example of a cell in each of the following stages: interphase, prophase, metaphase, anaphase, and telophase.

3. Draw the dividing cell in the appropriate area for each stage of the cell cycle, exactly as it appears. Include your drawings in Table 3.

 Onion Root Tip: 100X
 Onion Root Tip: 100X
 Onion Root Tip: 100X
 Onion Root Tip: 100X
 Table 1: Mitosis Predictions Predictions: Supporting Evidence:
 Table 2: Mitosis Data Number of Cells in Each Stage Total Number of Cells Calculated % of Time Spent in Each Stage Interphase: Interphase: Prophase: Prophase: Metaphase: Metaphase: Anaphase: Anaphase: Telophase: Telophase: Cytokinesis: Cytokinesis:
 Table 3: Stage Drawings Cell Stage: Drawing: Interphase: Prophase: Metaphase: Anaphase: Telophase: Cytokinesis:

Post-Lab Questions

1. Label the arrows in the slide image below with the appropriate stage of the cell cycle.

2. In what stage were most of the onion root tip cells? Based on what you know about cell cycle division, what does this imply about the life span of a cell?

3. Were there any stages of the cell cycle that you did not observe? How can you explain this using evidence from the cell cycle?

4. As a cell grows, what happens to its surface area to volume ratio? (Hint: Think of a balloon being blown up). How does this ratio change with respect to cell division?

5. What is the function of mitosis in a cell that is about to divide?

6. What would happen if mitosis were uncontrolled?

7. How accurate were your time predication for each stage of the cell cycle?

8. Discuss one observation that you found interesting while looking at the onion root tip cells.

Experiment 2: Tracking Chromosomal DNA Movement through Mitosis

Although mitosis and meiosis share similarities, they are different processes and create very different results. In this experiment, you will follow the movement of the chromosomes through mitosis to create somatic daughter cells.

 Materials 2 Sets of Different Colored Pop-it® Beads (32 of each - these may be any color) (8) 5-Holed Pop-it® Beads (used as centromeres)

Procedure

Genetic content is replicated during interphase. DNA exists as loose molecular strands called chromatin; it has not condensed to form chromosomes yet.

Sister chromatids begin coiling into chromosomes during prophase. Begin your experiment here:

1. Build a pair of replicated, homologous chromosomes. 10 beads should be used to create each individual sister chromatid (20 beads per chromosome pair). Two five-holed beads represent each centromere. To do this...

 Figure 5: Bead set-up. The blue beads represent one pair of sister chromatids and the black beads represent a second pair of sister chromatids. The black and blue pair are homologous.

a. Start with 20 beads of one color to create your first sister chromatid pair. Five beads must be snapped together for each of the four different strands. Two strands create the first chromatid, and two strands create the second chromatid.

b. Place one five-holed bead flat on a work surface with the node positioned up. Then, snap two of the four strands into the bead to create an “I” shaped sister chromatid. Repeat this step with the other two strands and another five-holed bead.

c. Once both sister chromatids are constructed, connect them by their five-holed beads creating an “X” shape.

d. Repeat this process using 20 new beads (of a different color) to create the second sister chromatid pair. See Figure 5 for reference.

2. Assemble a second pair of replicated sister chromatids; this time using 12 beads, instead of 20, per pair (six beads per each complete sister chromatid strand).

 Figure 6: Second set of replicated chromosomes.

3. Repeat this process using 12 new beads (of a different color) to create the second set of sister chromatids. See Figure 6 for reference.

4. Configure the chromosomes as they would appear in each of the stages of the cell cycle (prophase, metaphase, anaphase, telophase, and cytokinesis). Diagram the images for each stage in the section titled “Cell Cycle Division: Mitosis Beads Diagram”. Be sure to indicate the number of chromosomes present in each cell for each phase.

Cell Cycle Division: Mitosis Beads Diagram:

Prophase

Metaphase

Anaphase

Telophase

Cytokinesis

Post-Lab Questions

1. How many chromosomes did each of your daughter cells contain?

2. Why is it important for each daughter cell to contain information identical to the parent cell?

3. How often do human skin cells divide? Why might that be? Compare this rate to how frequently human neurons divide. What do you notice?

4. Hypothesize what would happen if the sister chromatids did not split equally during anaphase of mitosis.

Experiment 3: The Importance of Cell Cycle Control

Some environmental factors can cause genetic mutations which result in a lack of proper cell cycle control (mitosis). When this happens, the possibility for uncontrolled cell growth occurs. In some instances, uncontrolled growth can lead to tumors, which are often associated with cancer, or other biological diseases.

In this experiment, you will review some of the karyotypic differences which can be observed when comparing normal, controlled cell growth and abnormal, uncontrolled cell growth. A karyotype is an image of the complete set of diploid chromosomes in a single cell.

 Materials *Computer Access *Internet Access *You Must Provide

Procedure

1. Begin by constructing a hypothesis to explain what differences you might observe when comparing the karyotypes of human cells which experience normal cell cycle control versus cancerous cells (which experience abnormal, or a lack of, cell cycle control). Record your hypothesis in Post-Lab Question 1. Note: Be sure to include what you expect to observe, and why you think you will observe these features. Think about what you know about cancerous cell growth to help construct this information

2. Go online to find some images of abnormal karyotypes, and normal karyotypes. The best results will come from search terms such as “abnormal karyotype”, “HeLa cells”, “normal karyotype”, “abnormal chromosomes”, etc. Be sure to use dependable resources which have been peer-reviewed

3. Identify at least five abnormalities in the abnormal images. Then, list and draw each image in the Data section at the end of this experiment. Do these abnormalities agree with your original hypothesis? Hint: It may be helpful to count the number of chromosomes, count the number of pairs, compare the sizes of homologous chromosomes, look for any missing or additional genetic markers/flags, etc.

Data

1.

2.

3.

4.

5.

Post-Lab Questions

1. Record your hypothesis from Step 1 in the Procedure section here.

3. Suppose a person developed a mutation in a somatic cell which diminishes the performance of the body’s natural cell cycle control proteins. This mutation resulted in cancer, but was effectively treated with a cocktail of cancer-fighting techniques. Is it possible for this person’s future children to inherit this cancer-causing mutation? Be specific when you explain why or why not.

Pre-Lab Questions

1. Arrange the following molecules from least to most specific with respect to the original nucleotide sequence: RNA, DNA, Amino Acid, Protein

2. Identify two structural differences between DNA and RNA.

3. Suppose you are performing an experiment in which you must use heat to denature a double helix and create two single stranded pieces. Based on what you know about nucleotide bonding, do you think the nucleotides will all denature at the same time? Use scientific reasoning to explain why.

Experiment 1: Coding

In this experiment, you will model the effects of mutations on the genetic code. Some mutations cause no structural or functional change to proteins while others can have devastating affects on an organism.

Procedure:

1. Using the red, blue, yellow and green beads, devise and lay out a three color code for each of the following letters (codon). For example Z = green : red : green.

In the spaces below the letter, record your “code”.

 C: E: H: I: K: L: bbb ggg rrr yyy bgr Grb
 M: O: S: T: U: yrg yby byb Rgr Gyg
 Create codons for: Start: Stop: Space: bbr ggr yyr

2. Using this code, align the beads corresponding to the appropriate letter to write the following sentence (don’t forget start, space and stop): The mouse likes most cheese

a. How many beads did you use? 87

There are multiple ways your cells can read a sequence of DNA and build slightly different proteins from the same strand. We will not go through the process here, but as an illustration of this “alternate splicing”, remove codons (beads) 52 - 66 from your sentence above.

b. What does the sentence say now? (re-write the entire sentence) The mouse likes cheese

Mutations are simply changes in the sequence of nucleotides. There are three ways this occurs:

1. Change a nucleotide(s)

2. Remove a nucleotide(s)

3. Using the sentence from exercise 1B:

a. Change the 24th bead to a different color. What does the sentence say now (re-read the entire sentence)? Does the sentence still make sense?

The moose likes cheese

b. Replace the 24th bead and remove the 20th bead (remember what was there). What does the sentence say (re-read the entire sentence)? Does the sentence still make sense? If it doesn’t make sense as a sentence, are there any words that do? If so, what words still make sense?

The muse likes cheese

c. Replace the 20th bead and add one between bead numbers 50 and 51. What does the sentence say now? Does the sentence still make sense?

d. In 3.a (above) you mutated one letter. What role do you think the redundancy of the genetic code plays in this type of change?

e. Based on your observations, why do you suppose the mutations we made in 3.b and 3.c are called frame shift mutations?

f. Which mutations do you suspect have the greatest consequence? Why?

Experiment 2: Transcription and Translation

DNA codes for all of the proteins manufactured by any organism (including you!). It is valuable, highly informative and securely protected in the nucleus of every cell. Consider the following analogy:

An architect spends months or years designing a building. Her original drawings are valuable and informative. She will not provide the original copy to everyone involved in constructing the building. Instead, she gives the electrician a copy with the information she needs to build the electrical system. She will do the same for the plumbers, the framers, the roofers and everyone else who needs to play a role to build the structure. These are subsets of the information contained in the original copy. Your cell does the same thing. The “original drawings” are contained in your DNA which is securely stored in the nucleus.

Nuclear DNA is “opened up” by an enzyme called helicase, and a subset of information is transcribed into RNA. RNA is a single strand version of DNA, where the nucleotide uracil, replaces thymine. The copies are sent from the nucleus to the cytoplasm in the form of messenger RNA (mRNA ). Once in the cytoplasm, transfer RNA (tRNA) links to the codons and aligns the proper amino acids, based on the mRNA sequence. Protein builders called ribosomes float around in the cytoplasm, latch onto the strand of mRNA and sequentially link the amino acids together that the tRNA has lined up for them. This construction of proteins from the mRNA is known as translation.

Procedure

1. Use a pen or pencil to write a five word sentence using no more than eight different letters in the space below.

2. Now, use the red, blue, green, and yellow beads to form “codons” (three beads) for each letter in your sentence. Then, create codons to represent the “start, “space” and stop” regions within your sentence. Write the sentence using the beads in the space below:

3. How many beads did you use?

4. Assign one Pop-It® bead to represent each codon. You do not need to assign a Pop-It® bead for the start, stop and space regions. These will be your amino acids.

5. Connect the Pop-It® beads to build the chain of amino acids that code for your sentence (leave out the start, stop, and space regions).

6. How many different amino acids did you use?

7. How many total amino acids did you use?

Experiment 3: DNA Extraction

Much can be learned from studying an organism’s DNA. The first step to doing this is extracting DNA from cells. In this experiment, you will isolate DNA from the cells of fruit.

 Materials (1) 10 mL Graduated Cylinder (2) 100 mL Beakers 15 cm Cheesecloth 1 Resealable Bag 1 Rubber Band (Large. Contains latex; please wear gloves when handling if you have a latex allergy). Standing Test Tube Wooden Stir Stick *Fresh, Soft Fruit (e.g., Grapes, Strawberries, Banana, etc.) *Scissors **DNA Extraction Solution ***Ice Cold Ethanol *You Must Provide **Contains sodium chloride, detergent and water ***For ice cold ethanol, store in the freezer 60 minutes before use.

REMINDER: You are REQUIRED to video yourself performing steps 3 through 9 of the procedure below. You MUST submit the video with the lab to receive credit for this experiment.

Procedure:

1. If you have not done so, prepare the ethanol by placing it in a freezer for approximately 60 minutes.

2. Put pieces of a soft fruit into a plastic zipper bag and mash with your fist. The amount of food should be equal to the size of approximately five grapes.

3. Use the 10 mL graduated cylinder to measure 10 mL of the DNA Extraction Solution. Transfer the solution from the cylinder to the bag with the fruit it in. Seal the bag completely.

4. Mix well by kneading the bag for two minutes.

5. Create a filter by placing the center of the cheesecloth over the mouth of the standing test tube, pushing it into the tube about two inches, and securing the cheesecloth with a rubber band around the top of the test tube.

6. Cut a hole in the corner of the bag and filter your extraction by pouring it into the cheesecloth. You will need to keep the filtered solution which passes through the cheese cloth into the standing test tube.

7. Rinse the 10 mL graduated cylinder, and measure five mL of ice-cold ethanol. Then, while holding the standing test tube at a 45° angle, slowly transfer the ethanol into the standing test tube with the filtered solution.

 Figure 6: DNA extraction. The color has been enhanced by dying the fruit with a substance that glows under black light.

8. DNA will precipitate (come out of solution) after the ethanol has been added to the solution. Let the test tube sit undisturbed for 2 - 5 minutes. You should begin to see air bubbles form at the boundary line between the ethanol and the filtered fruit solution. Bubbles will form near the top, and you will eventually see the DNA float to the top of the ethanol.

9. Gently insert the stir stick into the test tube. Slowly raise and lower the tip several times to spool and collect the DNA. If there is an insufficient amount of DNA available, it may not float to the top of the solution in a form that can be easily spooled or removed from the tube. However, the DNA will still be visible as white/clear clusters by gently stirring the solution and pushing the clusters around the top.

Post-Lab Questions

1. What is the texture and consistency of the DNA?

2. Why did we use a salt in the extraction solution?

3. Is the DNA soluble in the aqueous solution or alcohol?

4. What else might be in the ethanol/aqueous interface? How could you eliminate this?

5. Which DNA bases pair with each other? How many hydrogen bonds are shared by each pair?

6. How is information to make proteins passed on through generations?

Pre-Lab Questions

1. In a species of mice, brown fur color is dominant to white fur color. When a brown mouse is crossed with a white mouse all of their offspring have brown fur. Why did none of the offspring have white fur?

2. Can a person’s genotype be determine by their phenotype? Why or why not?

3. Are incomplete dominant and co-dominant patterns of inheritance found in human traits? If yes, give examples of each.

4. Consider the following genotype: Yy Ss Hh. We have now added the gene for height: Tall (H) or Short (h).

a. How many different gamete combinations can be produced?

b. Many traits (phenotypes), like eye color, are controlled by multiple genes. If eye color were controlled by the number of genes indicated below, how many possible genotype combinations would there be in the following scenarios?

5 Eye Color Genes:

10 Eye Color Genes:

20 Eye Color Genes:

Experiment 1: Punnett Square Crosses

In this experiment you will use monohybrid and dihybrid crosses to predict patterns of inheritance.

Procedure:

Part 1: Punnett Squares

1. Set up and complete Punnett squares for each of the following crosses: (remember Y = yellow, and y = blue)

 Y Y and Y y Y Y and y y

2. What are the resulting phenotypes?

3. Are there any blue kernels? How can you tell?

4. Set up and complete a Punnett squares for a cross of two of the F1 from Step 1 (above).

5. What are the genotypes of the F2 generation?

6. What are their phenotypes?

7. Are there more or less blue kernels than in the F1 generation?

8. Identify the four possible gametes produced by the following individuals:

 a) YY Ss: ______ ______ ______ ______ b) Yy Ss: ______ ______ ______ ______ c) Create a Punnett square using these gametes as P and determine the genotypes of the F1:

What are the phenotypes? What is the ratio of those phenotypes?

Part 2 and 3 Setup

1. Use the permanent marker to label the two 100 mL beakers as “1” and “2”.

2. Pour 50 of the blue beads and 50 of the yellow beads into Beaker 1. Sift or stir the beads around to create a homogenous mixture.

3. Pour 50 of the red beads and 50 of the green beads into Beaker 2. Sift or stir the beads around to create a homogenous mixture.

Assumptions for the remainder of the experiment:

· Beaker 1 contains beads that are either yellow or blue.

· Beaker 2 contains beads that are either green or red.

· Both beakers contain approximately the same number of each colored bead.

· These colors correspond to the following traits (remember that Y/y is for kernel color and S/s is for smooth/wrinkled):

1. Yellow (Y) vs. Blue (y)

2. Green (G) vs. Red (g).

Part 2: Monohybrid Cross

1. Randomly (without looking) take two beads out of Beaker 1. This is the genotype of Individual #1. Record the genotype in Table 1. Do not put these beads back into the beaker.

 Table 1: Parent Genotypes: Monohybrid Crosses Generation Genotype of Individual 1 Genotype of Individual 2 P P1 P2 P3 P4

2. Repeat Step 1 for Individual #2. These two genotypes represent the parents (generation P) for the next generation.

3. Set up a Punnett square and determine the genotypes and phenotypes for this cross. Record your data in Table 2

4. Repeat Step 3 four more times (for a total of five subsequent generations). Return the beads to their respective beakers when finished.

 Table 2: Generation Data Produced by Monohybrid Crosses Parents Possible Offspring Genotypes Possible Offspring Phenotypes Genotype Ratio Phenotype Ratio P P1 P2 P3 P4

Post-Lab Questions

Part 2: Monohybrid Cross

1. How much genotypic variation do you find in the randomly picked parents of your crosses?

2. How much in the offspring?

3. How much phenotypic variation?

4. Is the ratio of observed phenotypes the same as the ratio of predicted phenotypes? Why or why not?

5. Pool all of the offspring from your five replicates. How much phenotypic variation do you find?

6. What is the difference between genes and alleles?

7. How might protein synthesis execute differently if a mutation occurs?

8. Organisms heterozygous for a recessive trait are often called carriers of that trait. What does that mean?

9. In peas, green pods (G) are dominant over yellow pods. If a homozygous dominant plant is crossed with a homozygous recessive plant, what will be the phenotype of the F1 generation? If two plants from the F1 generation are crossed, what will the phenotype of their offspring be?