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BIO322: Genetics

Laboratory Manual

Department of Biology

Wilmington College of Ohio

Spring 2011

Douglas J. Burks, Ph.D.

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Table of Contents

Section Page

Goals and Objectives … 3

Assessment Methods … 3

Laboratory Polices … 4

Evaluation … 5

Laboratory 1: Independent Segregation … 6

Laboratory 2: Meiosis … 12

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BIO350: Topics – Molecular Pharmacology

Douglas Burks

Jim Reynolds

Department of Biology

Wilmington College of Ohio

Laboratory Manual

Goals and Objectives

We have set several goals for you this semester in the laboratory of molecular

pharmacology. These include:

1. to develop scientific questions in a way that lead to useful hypotheses,

design experiments to address the question, perform the experiments,

critically evaluate data, and analyze results of experiments;

2. to develop basic laboratory skills involved in doing modern genetics

research;

3. to enhance your ability to undertake a primary literature in developing

background and information for addressing scientific questions in the

laboratory;

4. and to strengthen written communication skills.

Assessment Methods

We will use several different methods to assess your growth and progress in the

laboratory. These include:

1. pre-lab quizzes on preparation for laboratory;

2. observation of your work at the lab bench;

3. research proposals;

4. analysis of data;

5. maintaining an accurate and current laboratory notebook;

6. and preparing research papers which include primary literature, results

and analysis of experimental results.

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Laboratory Policies

1. Laboratory personal are expected to follow the safety policies of the Biology

Department that are found in the BIO231 Laboratory Manual.

2. Students are expected to come to the laboratory prepared to undertake

research which includes doing any reading of necessary materials before

coming to lab.

3. Students may miss one laboratory period without loss of points towards their

final grade. After one absence students will be assessed -5% of the

laboratory grade per absence. Students are not excused from any

assignment for any laboratory experiment.

4. All late assignments will be assessed a penalty for each day it is late as

described in the Biology Writing Manual. The late policy states:

a. Wilmington College biology professors have adopted a unified policy

concerning late assignments. An assignment is late any time after 4:00

PM on the assignment’s due date, unless otherwise indicated. In

addition, any incomplete, substandard assignment submitted at the

deadline just to “get something in” will be considered late.

b. Submission of the final assignment after the deadline will result in a

20% deduction of points for each working day it is late. For example,

if you hand in your late assignment within one working day of the due

date, the highest score you can possibly receive is an 80%. If you

hand in your late assignment within two working days of the due date,

the highest score you can receive is a 60%. If you hand in your

assignment within three working days of the due date, the highest

score you can receive is a 40%. Late assignments will not be accepted

after three working days. (http://plato.wilmington.edu/faculty/dburks/Writing%20Manual%20August%202010.pdf)

This policy will be strictly enforced.

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Grade Evaluation for Laboratory

Percent of Laboratory Grade

Pre-lab quizzes 10%

Observation of Laboratory Work 10%

Laboratory Notebook 25%

Laboratory Reports 55%

Total 100%

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LABORATORY 1

TESTING A HYPOTHESIS

Corn Seed Color

3:1

9:3:3:1

Background:

Seed Color in corn is variable. One gene that affects color has two

alleles. For the color gene one allele is for yellow and one allele for

purple for this color gene. Purple color is dominant to yellow which is

recessive. A second trait is for seed shape. One allele for seed shape is

smooth which is dominant to a wrinkled kernel which is recessive.

In this experiment we want to test if the gene alleles for color

show independent segregation and if the two genes show independent

Name: ______________________

Date: _______________________

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assortment. A monohybrid cross for independent segregation should

give a 3:1 ratio.

Monohybrid Cross in corn

Trait Dominant

allele

Recessive

allele

Seed color

Seed Shape

Ratio

Dihybrid Cross in corn

Trait D seed

color D

seed

shape

D seed

color R

seed

shape

R seed

color D

seed

shape

R seed

color R

seed

shape

Phenotype

Ratio

To test independent segregation and assortment we have corn cob

samples that you can count the number of seeds showing various seed

types from monohybrid and dihybrid crosses. You can calculate ratios

from those samples.

Once you have those numbers how do we judge if they are the expected

ratio. It is doubtful that you will get exactly 3:1 or 9:3:3:1. How close is

close enough to say that an experimental number is the expected ratio.

The answer is to use a statistical test. The statistical test is the Chi

Square Test. The Chi Square is a statistical test used to judge the

goodness of fit of an observed distribution to a theoretical distribution.

In our case the theoretical distribution is 3:1 and 9:3:3:1. In this

statistical test we are testing the null hypothesis. The null hypothesis

is that the numbers are different due to chance.

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Chi Square is

We can look at an example in pea plants. When we do the cross

between two hybrid tall plants (Tt) we expect to see a 3 tall: 1 dwarf

plant ratio. We do the cross and plant 1000 seeds and observe 739 tall

plants and 261 dwarf plants. We expected theoretical numbers of 750

tall and 250 dwarf plants. Is this an example of independent

segregation or not. Let’s do a Chi Square Test.

Observed Expected (O-E) (O-E)2

Tall 739 750 -11 121

Dwarf 261 250 11 121

(O-E)2/expectd

Tall

121/150 .1637

(O-E)2/expectd

Tall

121/750 .860

X2 ∑(O-

E)2/expected

.970

Degrees of freedom is the number of classes – 1. df = n-1. In our case

here we have two classes (dominant and recessive) and therefore df = 2-

1 = 1.

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The p value from a p calculator is 0.327. We reject the null hypothesis

that the numbers are different by chance (they are the same).

(http://www.danielsoper.com/statcalc/calc11.aspx )

Experiment

You are to test three hypotheses in this experiment.

1. Purple vs. yellow seed (independent segregation; 3:1 ratio from

monohybrid cross).

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2. Smooth vs. wrinkled seed (independent segregation; 3:1 ratio from

monohybrid cross).

3. Smooth vs. wrinkled; purple vs. yellow (independent assortment;

9:3:3:1 ratio from dihybrid cross).

For each experiment you are to count all the seed on one cob and

calculate Chi Square and a p value.

Monohybrid Cross: Corn Kernel Coloration

Number of Purple

Seeds

Number of Yellow

seeds

Actual Number

Actual Ratio

Expected Number

Expected Ratio

Monohybrid Cross: Corn Kernel Shape

Number of Smooth

Seeds

Number of Wrinkled

seeds

Actual Number

Actual Ratio

Expected Number

Expected Ratio

Dihybrid Cross: Corn Kernel Coloration and Shape

# Purple

Smooth

# Purple

Wrinkled

# Yellow

Smooth

# Yellow

Wrinkled

Actual

Number

Actual

Ratio

Expected

Number

Expected

Ratio

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Show calculation of Chi Square for each.

Determine the p value for each

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

Do the alleles show independent segregation and do the two genes show

independent assortment?

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

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__________________________________________________________________

__________________________________________________________________

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LABORATORY 2

MEIOSIS

Background

Meiosis is the cellular division that occurs in germ cells that leads to a reductive

division and the production of gametes for sexual reproduction. Chromosomes, the units

of Mendelian segregation and assortment, are randomly divided and given to gametes in

this process. Homologous chromosomes pair and separate during the first division of

meiosis leading to the reductive division (2n � 1n) (Figure 1). It is also during

meiosis 1 that crossing-over occurs leading to new linkage patterns in genes within a

homologous pair of chromosomes. In meiosis II duplicated chromosomes separate sister

chromatids leading to 1n cells containing unduplicated chromosomes that are ready for

fertilization. Because of crossing-over the daughter cells that result from meiosis II are

not identical (Figure 2).

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The lilium anther and pollen production is a classic model system to study meiosis

by microscopy. Meiosis in plants occurs in structures called sporangia. The sporangia of

flowering plants are located in the flower. Flowers have both male and female sporangia.

The male sporangium is called the anther and the female sporangium is an ovule. Haploid

microspores are produced inside the pollen sacs by meiosis, and they usually are in

tetrads (groups of four). In lilium there are four pollen sacs (microsporangia) per anther.

In the center of each pollen sac are tetrads of microspores. A tetrad is simply a group of

four. Why are the microspores in groups of four? The tetrads are surrounded by the

tapetum, a nutritive tissue derived from the innermost layer of the pollen sac (Figure 3).

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In this laboratory you will undertake a study of meiosis in lilium anthers (Figure 4).

Protocol

Identifying meiotic stages.

1. Obtain a set of lilium meiosis slides and a compound light microscope.

a. Review how to use a microscope

b. Review using an oil immersion lens

i. Focus very carefully with the 40x objective over the stained

specimen on the slide.

1. Once focused, do not alter focus for the next three steps!

ii. Rotate turret half way so that the 40x and 100x objectives straddle

specimen.

iii. Apply a small drop of oil directly on the slide over the specimen.

iv. Rotate 100x objective into the immersion oil

v. Never get oil on any other lens.

1. Never go back to the 10x or 40x objectives after you

have applied oil to the specimen since oil can ruin the

lower power objectives

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vi. Clean up all oil when finished. 1. When you have finished for the day, wipe the 100x oil

immersion objective carefully with lens paper to remove all

oil. Wipe oil from the slide thoroughly with a Kimwipe.

Cleanse stage should any oil have spilled on it. Recap the

immersion oil container securely, replace in drawer.

2. Focus on anther with the 4X lens

3. Exam the tissue at 40X and learn landmarks of tissue

4. Exam the tissue at 100X and find and identify each stage of meiosis.

Photographing meiotic cells.

1. Obtain a set of lilium meiosis slides and a compound light microscope.

2. Focus on the lilium tissue at 4X

3. Obtain a photomicroscopy camera.

4. Log onto the computer.

5. Carefully insert camera into eyepiece and insert the USB cord into the computer.

6. Launch the microscopy software.

7. Place your USB drive into the computer

8. Photograph the tissue at 4X, 10X, and 40X

9. At 100X photograph each stage of meiosis.

10. Transfer images to your USB drive

11. Erase file on microscopy software.

Report

You are to write a description of meiosis in lilium in which you describe each stage of

meiosis using your photographs of stages as figures to illustrate your description. The

report should have a general background on the structure of the reproductive organs in

lilium and on meiosis. It should then have a detailed description of each stage of meiosis.

Reports should be typewritten, Figures should be placed within text and all figures

should have a figure legend (in a font smaller than text).

The report is due @ 1:00 PM on February 1, 2011. Late policy

applies.

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