MANAGEMENT & SCIENCE

UNIVERSITY

CENTRE FOR FOUNDATION STUDIES

BASIC GENETICS
FGS0054

LABORATORY MANUAL

Name : _______________________________________

Group :_____________________________

1
 CONTENT

NO PRACTICAL PAGE
Laboratory Safety 3
1 Isolating DNA From the Cells 4
2 Cell Division 6
3 Mendel’s Genetic 8
4 Karyotyping 11

2
 LABORATORY SAFETY

Below are a few guidelines to conduct a practical exercise properly and safely.

a) Students can only be in the laboratory with the presence of a lecturer/tutor

assigned.
b) Please do not run, play around or do any indecent behavior while in the lab.
c) In the case of an accident, injury or illness immediately inform the lecturer
or tutor in the laboratory.
d) Please do not eat, drink, smoke or handle contact lenses in the laboratory.
e) Be aware of the location and operation of all emergency equipment and how
to call for help if needed.
f) Know the potential hazards, precautions and safety procedures before
conducting a practical exercise.
g) Please wear comfortable, inexpensive clothing and most importantly a LAB
COAT.
h) Please wash your hands after handling chemicals, animals or after
removing your gloves.
i) Please do not intentionally sniff any chemicals.
j) Please promptly report any faulty equipment, damage specimens, water or
gas leaks to the lecturer or tutor in the laboratory.
k) Ask the lecturer or tutor if you are unsure of any part in the practical
procedure before the practical starts.
l) Please clean work surfaces, switch off all electrical outlets after each
practical exercise.
m) If there is any fire or a fire alarms rings, immediately leave the lab in an
orderly manner to the designated safe area.

3
 PRACTICAL 1

TITLE : ISOLATING DNA FROM THE CELLS

OBJECTIVE:

To isolate DNA from kiwi fruits cells.

INTRODUCTION:

Deoxyribonucleic acid (DNA) is the genetic material of all living organisms and
some viruses. The genetic material of prokaryotes is double-stranded DNA
localized into one or a few chromosomes. Typically, prokaryotic chromosomes
are circular, but linear chromosomes are found in a number of species.
Prokaryotic genomes consist mostly of unique DNA sequence. They have only a
few repeated sequence and genes.

MATERIALS:

1. Kiwi fruits 12. Detergent-mix

2. Mortar &pestle 13. 95% Alcohol
3. Beaker 500 ml 14. Ice cubes
4. Test tube 15. Tissue
5. Test tube rack 16. Weighing meter
6. Bacterial loops/wire loop 17. Weighing plastic
7. Measuring cylinder (10 ml) 18. Filter gauze
8. Cover slip 19. Glass rod
9. Microscope 20. Spatula
10. Water bath 21. Sodium chloride
11. Gloves 22. Knife/Razor blade

4
PROCEDURES:

1. Prepare the extraction buffer by mixing together 3g of salt, 10ml detergent

and 100ml water in a beaker. Stir slowly until the salt has dissolved,
without producing the bubble/foam.
2. Add 10ml of chilled alcohol in a test tube and place the test tube into a
beaker/box containing ice cube.
3. Cut a kiwi fruit into half size and mash it into small chunks using mortar
and pestle.
4. Transfer the squashed kiwi and put them into a beaker containing the
extraction buffer.
5. Incubate the kiwi and extraction buffer in the water bath (60˚C) and leave
for 15 minutes.
6. After 15 minutes, filter the kiwi mixture into a beaker using filter gauze.
7. Fill the test tube about half full of the kiwi extract and carefully add the
cold alcohol by pouring it slowly (at flat angle) down the side of the test
tube.
8. Both of the liquids are to make sure that they do not mix but the alcohol
builds a separate layer on top of the fruit sap.
9. Place the test tube in a test tube rack and observe it. A white jelly-like
substance (DNA) begins to form between the alcohol and the fruit sap after
a short time.
10. Scoop out the DNA filament using the wire loop and place it on a specimen
slide.

DISCUSSIONS:

• What was the purpose of the detergent?

• What was the purpose of the salt?
• Why was it necessary to mash the fruit?
• Why do you heat the mixture?
• What happen when cold alcohol is added?
• Would it make a difference if warm alcohol was added?

5
 PRACTICAL 2

TITLE: CELL DIVISION – Mitosis for Onion Root

OBJECTIVE:

1. To prepare a slide from onion root

2. To understand and determine the mitosis concepts and stages in onion
root processes
3. To differentiate the chromosome location and chromatin in mitosis and
meiosis phase

INTRODUCTION:

Mitosis is a process of nuclear division in which replicated DNA molecules of each

chromosomes are partition into two nuclei. Mitosis accompanied by cytokinesis.
It is a process which a dividing cell split in two, one partition the cytoplasm into
two nuclei.

Mitosis is divided into 5 steps: Interphase, prophase, metaphase, anaphase and

telophase. In interphase chromosome begin to split into two and the microtubules
radiates but forming an aster. Centrioles are not present in plants. Plants have
less distinct spindle pole organizing centers.

MATERIALS:

1. Sterile water 10. Petri plate

2. Microscope 11. Toluidine solution
3. Needle 12. Bunsen burner
4. Onion root 13. Tissue
5. Glass slide 14. Watch glass
6. Knife/Razor blade 15. Cover slip
7. Plastic dropper 16. Wooden holder
8. Glove 17. Matches
9. 70% alcohol

6
PROCEDURES:

1. Prepare the onion root around one week before experiment and place it in 8’
hydroxyquinoline which is a concentrated solution and also in ethanol/lactic
acid in the ratio 3:1. Then, soak the root for 3 to 4 hours. This process will
stop the cell division in the root of the onion cell. Shortly after, the root shifts
to a 70% alcohol solution.
2. Cut a small piece of onion root and place it on a watch glass.
3. Sprinkle 2 to 3 drops of sterile water on top of the root.
4. Heat the specimen for a while (not boil) and then keep the root until it cool.
5. Cut the root into smaller pieces and place it on the slide.
6. Drop a drop of toluidine solution on the specimen and heat it mildly near 3 to
4 times and cool it.
7. Smear the tissue of root and keep on top of a glass slide by using a needle.
8. Close the abstract of tissue with cover slide and press it with thumb and a
piece of paper to spread the organelles.
9. Observe the slide under the microscope.
10. For a better result, compare it with a prepared slide provided by lecturer.

DISCUSSIONS:

• What is the purpose of blue toluidine?

• Why should the onion root be crushed into small pieces?
• What is the purpose of pressing the specimen with a piece of paper?
• Why must we sprinkle few drops of sterile water on top of the onion root?

7
 PRACTICAL 3

TITLE: MENDELIAN GENETICS – CHI SQUARED TEST (X2)

OBJECTIVE:

To determine that the data from the appropriate experiment will have the same
ratio as theory.

INTRODUCTION:

Chi square test usually use to determine that data from experiment about
the same or near to theory. It is used to determine whether there is a good fit
between the observed and the expected data. It is also used to determine whether
the deviation id significant by chance. Generally, small deviation can be obtained
by chance while big deviation could not.

Formula for chi squared test;

X2 = (O-E)2
E
O = Number of individuals representing a particular phenotype
E = Number of expected individuals showing the phenotype
X2 = the sum of possible value (O-E)2 for different phenotypes

If p value more than 0.05 (p>0.05) this show that hypothesis F2 has 3:1
(monohybrid cross) ratio and 9:3:3:1 (dihybrid cross) ratio accepted.

EXAMPLE

Crossing between a tall green pea plants with a short plant produces tall plants
in the F1 generation. F1 plants are self-pollinated and produce 94 tall plants and
36 short plants in the F2 generation. Are the data in F2 fit the 3:1 ratio the
monohybrid crossing?

8
SOLUTION
Eg.
Phenotype Genotype O E (O-E) (O-E)2 (O-E)2 / E
Tall TT/Tt 94 97.5 -3.5 12.25 0.126
Short tt 36 32.5 3.5 12.25 0.377
X2 = 0.503
What is meant by X2 = 0.503 in the table above?

If the observed value (O) is the same as the expected theoretical value (E), the
value of X2 is zero. Therefore, high accuracy is obtained. The small value of X2
means that the observed data and the expected data are almost similar while the
bigger value of X2 shows that the deviation of the observed data (O) compared to
the expected data (E) is big.

How can we determine whether the deviation is within the expected limit if the
deviation is due to chance?

The limit for occurrence by chance set by statisticians is in 1 in 20 (probability =

0.05). This becomes the critical value for accepting or rejecting the result.

Chi-squared value is read from the chi-squared table.

Chi-squared table

9
The top row represents probability (P) while the first column on the left represents
degree of freedom (N). The degree of freedom is one less than the number of
phenotypes. In the above example, the number of phenotypes is two (2), tall and
short. Therefore, the degree of freedom is 2-1=1. In the table above, X2 = 0.503
is between the column 0.10 (X2 = 2.706) and 0.50 (X2 = 0.455). This means that,
if the same experiment is repeated 100 times, X2 = 0.503 will be obtained by
chance in 10 to 50 times. The probability obtained shows that the number
observed (O) is in good fit with the number expected (E).

MATERIALS:

Blue card Yellow card

Green card Red card
Plastic bag / 500 ml beaker

PROCEDURES:

Monohybrid Cross

1. Prepare 40 blue cards and 40 yellow cards. The blue card will represent T allele
and the yellow card will represent t allele.
2. Mix all cards in a plastic bag.
3. Take out two cards from the plastic bag and determine the combination i.e.
TT, Tt or tt.
4. Repeat step 3 until all 40 pairs of the cards are drawn.
5. Record all the combination in Table 1, and then count the X2 value.

Phenotype Genotype O E (O-E) (O-E)2 (O-E)2 / E

Tall TT/Tt
Short tt
X2 =

10
Dihybrid Cross

1. Mix 40 blue cards and 40 yellow cards in one plastic bag. Then mix 40 green
cards and 40 red cards in another plastic bag.

Blue card - Allele for tall (T)

Yellow cards - Allele for short (t)
Green card - Allele for purple flower (A)
Red card - Allele for white flower (a)

2. Take out 2 cards from each bag and determine the combination.
3. Repeat step 2 until you get 40 combinations.
4. Record all combinations in Table 2 and count the X2 value.

Phenotype Genotype O E (O-E) (O-E)2 (O-E)2 / E

Tall, purple
flower
Tall, white flower
Short, purple
flower
Short, white
flower
X2 =

DISCUSSION:

Do the monohybrid and dihybrid ratios obtained, fit the expected

Mendelian ratios?

11
 PRACTICAL 4

TITLE: KARYOTYPING

OBJECTIVE:

To determine gender and identify the abnormalities of human by using the

karyotype analysis.

INTRODUCTION:

Karyotype is the complete set of chromosomes in eukaryotic cell. Numbers and

types of karyotypes are specific traits of certain species. Human female has
44+XX and human male has 44+XY. Every chromosome has certain
characteristics such as size, location of the centromere and presence of other
structures like satellite and secondary constriction.

Chromosome identification in human cell is usually derived from actively

replicating white blood cell in culture in situ. Colcemid, a derivation of colchicine
is used to stop chromosome at metaphase and causes them to be short and fat.
Karyotype is prepared by cutting chromosomes shapes from the cell picture and
find their pairs which often in the same size. The chromosomes pairs will then be
identified and are arranged accordingly to particular groups.

MATERIALS:

Scissor Glue
Handout A4 papers

12
PROCEDURES:

1. Scatter chromosomes from the handouts, cut and arrange the chromosome
accordingly into particular chromosomes number, based on certain criteria.

Chromosome Characteristic
no.
1 The biggest chromosomes with centromere at the centre
2 Almost as big as the chromosomes 1 but the centromere at half of
the centre
3 Smaller than chromosomes 1 and 2 with centromere at half of the
centre
4-5 Hard to differentiate both chromosomes but usually chromosome 4
is longer than chromosome 5
6 Longer than chromosome X. It has centromere at half of the centre
7 Smaller than chromosome X with centromere at half of the centre
8 Centromere at half of the centre
9 Might be longer than chromosome 8 and often has colorless area at
the centre
10-12 Can be arranged according to size
13 Medium in size with centromere at the end and has no satellite
14 Shorter than chromosome 13 and has no satellite
15 Darker short arm and has no satellite
16 The biggest among chromosomes 16-19 and has centromere at the
centre
17 Its centromere is not far from half the centre
18 Smaller than chromosome 17 and has centromere about at the centre
19-20 Hard to differentiate, small and has centromere at the
centre
21 Very small, it has centromere at the end and has certain part that
carries satellite
22 Has satellite with darker short arm
Y Sometimes longer than chromosomes 21 and 22, has no satellite

2. The gender and abnormalities are determined from the arranged

chromosomes.

DISCUSSION:

Discuss all the abnormalities

13