Biology Urgent Assignment 21

Genetics: Experiment Instructions (Demonstration)

LEARNING GOALS

By the end of this unit, you should be able to do the following:

1. Explain the purpose of each step of isolating DNA from an onion.

2. Explain the difference between genotype and phenotype.

3. Explain Mendel's Law of Segregation including the concepts of having a pair of alleles, dominance, and passing on only one of the pair.

4. Explain Law of Independent Assortment focusing on the presence two different genes,

each with a pair of alleles.

5. Use a Punnett square to predict genotypes and phenotypes of offspring given parental genotype.

MATERIALS NEEDED (DNA Isolation)

Salt

Distilled water (not spring, mineral or tap water; often found in automotive section)

Straw

Coffee Filter

Bowl with Ice

Blender

Onion

Baking Soda

Coffee stirrer (get one for free at any fast food place)

Chilled rubbing alcohol (or liquid hand sanitizer)

Food Coloring (any color)

DNA ISOLATION

**You will be quizzed on the purpose of each step. Please pay attention to the information below!**

When isolating DNA, four key things need to occur. Firstly, the onion cells must be broken apart from each other by blending. This also breaks the cell walls in many places. Secondly, the plasma membrane and nuclear envelope must be removed. Since they are made of lipids, the dishwashing detergent can "wash" them away. Thirdly, the proteins wrapping the DNA need to be denatured. (Remember that denaturing a protein is changing its shape to make it inactive.) The salt in the buffer will cause the proteins to denature some, separating them from the DNA. Finally, the water-soluble DNA must be forced out of solution. Rubbing alcohol pulls on the DNA away from the water, preventing the DNA from staying dissolved. Even though the alcohol pulls on the DNA, it won't actually dissolve the DNA either, leaving DNA trapped between the water and the alcohol.

1. Make a Buffer solution by pouring 4 oz. of Distilled Water into a clean glass. Add ¼ teaspoon of Salt. Add 1 teaspoon of Baking Soda. Add 1 teaspoon of liquid detergent. Stir all ingredients well then chill in a bowl of ice water until thoroughly chilled.

2. Finely chop onion (or other food stuff) and add to a blender or food processor. Add a small amount of water to the onion then pulse in 10 sec. burst to break up the onion cells.

3. Place a teaspoon of mixed vegetable mush into a clean glass then add 2 teaspoons of the chilled buffer solution (from step # 1). Stir vigorously for at least 2 minutes.

4. Strain material from # 3 through a coffee filter to remove most of the plant refuse.

5. From the material filtered (filtrate), pour off the solid floating on the surface.

6. To extract the DNA from the solution, dip a drinking straw into the chilled rubbing alcohol then place the pad of your finger on the other end of the drinking straw which will keep the alcohol in the straw until you transfer it to the solution in the glass.

7. Release the alcohol in the straw very slowly by allowing it to trickle down the side of the glass containing the DNA solution. The alcohol being less dense than the buffer will float on top.

8. Where the two layers meet, you will be able to see a gelatinous sludge which will be the DNA. Gently insert the stirring stick through the layer of alcohol (barely penetrating the alcohol but not the buffer solution) to pick up the sludge.

9. Gingerly twirl the stirring stick back and forth with the tip of the stirring stick suspended just below the boundary between the alcohol and buffer solution.

After a minute of twirling, pull the stirring stick with the DNA attached up through alcohol. The DNA will will appear as a viscous clear sludge clinging to the stirring stick.

You have now successfully extracted DNA. Paste a picture of your DNA sludge into the worksheet (or add it to the Assignment Box directly) and answer questions about what each chemical did during this process!

GENETICS SIMULATION

You will be testing the Law of Segregation using eye color and then again using the presence of a tail. You will then test the Law of Independent Assortment using both eye color and tail at the same time.

Experiment #1 - Segregation of Eye Color (Black vs White)

1. Open the next two items in the module; a link to the Heredity I simulator and a worksheet. Return here for further instructions.

2. These little creatures in the simulation are called Flugals. Female Flugals are brown and smooth. Male Flugals are red and hairy. These differences are just there to distract you! The real experiment is about eye color.

3. Choose a mating pair of Flugals where one has white eyes and the other has black. It will also help if both have tails or both are missing tails. It doesn’t matter which, we just want to be able to ignore the tail and focus on the eyes.

4. The Flugals running around the screen now are the offspring from this first cross (F1). Thankfully, you don’t have to count them, because the number of each phenotype is shown at the top. You now have the information you need to answer the first three questions of Experiment #1. Go answer them now, including the hypothesis for what you think the next generation will look like.

5. Now click the pair of Flugals waiting at the bottom of the screen where it says “Click to select F1 cross.” This will create the F2 generation.

6. Compare your data to your hypothesis. Were you correct? Select “Click to Return” to reset the simulator.

Experiment #2 - Segregation of Tail and Tailless

Repeat the experiment above, but this time select parents with the same eye color, but different tails. Use the F1 results to determine which phenotype is dominant and the F2

results to determine the proportion of each phenotype. You should add the male and female results together when counting phenotypes.

Experiment #3 - Independent Assortment of Eye Color and Tail

The Law of Independent Assortment states that a fly should be able to pass on eye color and tail length irrespective of each other. Repeat the experiment, but this time select one parent that has black eyes and no tail, and the other parent with white eyes and a tail present. Mate these together for two generations and then look at the data. (Remember to add both sexes together.)

The worksheet will focus your math on looking at both traits individually (again) and both traits together.

Conclusions - The flies in the first generation had alleles for red eye/wings from their mother and sepia eye/apterous from their father. If they passed on random combinations of eye and wing alleles to the following generation, the ratios should come out with 0.56, 0.19, 0.19, and 0.06. Did they occur as expected? You can make either a 4 x 4 Punnett square to describe this or you can do the multiplication of two individual Punnett squares (see prelab example of tall plants with purple flowers).

NEXT STEP

This module includes an Assignment Box for your worksheet and also a Quiz. You should be prepared to answer questions about the patterns in the data that you saw.