CENTRE FOR FOUNDATION

STUDIES
BASIC GENETICS
FGS0054
LABORATORY MANUAL

Name :
Group :
Lecture name:

CONTENT

NO
1
2
3
4
5

PRACTICAL
Laboratory Safety
Isolating DNA From the Cells
Cell Division
Mendels Genetic
Karyotyping
Micropipetting

PAGE
3
4
6
8
11
14

LABORATORY SAFETY

Below are a few guidelines to conduct a practicle 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 behaviour 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 practicle 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 speciments, 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 practicle
procedure before the practicle starts.
l) Please clean work surfaces, switch off all electrical outlets after each
practicle 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.

PRACTICAL 1: ISOLATION OF DNA

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. Mortar & pastle
2. Beaker 500 ml
3. Test tube
4. Test tube rack
5. Bacterial loops / wire loop
6. Filter paper
7. Plain slide & cover slip
8. Microscope
9. Water bath (600C)
10.Kiwi fruits
11.Self-detergent mix
12.Alcohol 95%
13.Ice cube

PROCEDURES:
1. A solution is made up from 3g salt, 10ml washing up liquid (not soap)
and 100ml water in a beaker. It is then stirred thoroughly to dissolve
the salt completely without producing the foam.
2. 10ml of chilled alcohol is added in a test tube and the test tube is
placed into a beaker containing ice cube.
3. A kiwi fruit is peeled and chopped and mashed in a mortar.
4. The squashed kiwi was scooped into a beaker and 100ml of the saltdetergent mix is added.
5. The beaker is placed in the water bath (60C) and left for 15 minutes.
6. The mixture is placed into Buncher funnel after 15 minutes and the
liquid is collected in a test tube. Half tube of the liquid is collected.
7. The cold alcohol is drizzled very carefully (at flat angle) into the test
tube that contains the kiwi extract. Both of the liquids were to make

sure that they do not mix but the alcohol builds a separate layer on top
of the fruit sap.
8. The test tube is then placed in a test tube rack and observed it. A
white layer begins to form between the alcohol and the fruit sap after a
short time. The DNA filaments in the white layer were observed.
9. The filament was scooped out with the wire loop and placed on a slide.
10.The DNA filament was observed under the microscope.

QUESTIONS:
1.
2.
3.
4.
5.
6.

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 and cool the mixture?
What happen when cold alcohol is added?
Would it make a difference if warm alcohol was added?

PRACTICAL 2: CELL DIVISION

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
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:
Sterile water
Microscope
Needle
Onion root
Slide

Alcohol solution
Petri plate
Toluidin solution
Bunsen burner

PROCEDURES:
1. Prepare the onion root around one week before experiment and place it in
8 hydroxyuinoline 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 form onion root and place it on top of Petri plate.
3. Sprinkle 2 to 3 drops of sterile water on top of the root.
4. Heat the specimen 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 few drops of toluidin solution on the specimen and heat it mildly
near 3 to 4 times and later cool it.
7. Smear the tissue of root and keep on top of a slide by using a needle.
8. Close the abstract of tissue with tiny slide and press it with thumb and a
piece of paper to spread the organelles.
9. Observe the slide under a microscope.
10.For a better result, compare it with a sample slide provided by lecturer.

QUESTIONS:
1.
2.
3.
4.

What is the usage of blue toluidin?

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?

PRACTICAL 3: MENDELIAN GENETICS

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:

2.

1. 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 = (OE)2
E
O
E
X2

= Number of individuals representing a particular phenotype

= Number of individuals expected to show that phenotype
= the sum of possible value (O-E)2 for different phenotypes

3. 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?
SOLUTION
Phenoty
pe
Tall
Short

Genoty
pe
TT
tt

(O-E)

94
36

97.5
32.5

-3.5
3.5

(O-E)2

(O-E)2 /
E
12.25
0.126
12.25
0.377
2
X = 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
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 phenotype is
two (2), tall and short. Therefore the degrees of freedom is 2-1=1. In the
table above, X2 = 0.503 is between the column 0.30 (X2 = 1.07) and 0.50 (X2
= 0.46). this means that, if the same experiment is repeated 100 times, X2 =
0.503 will be obtained by chance in 30 to 50 times. The probability obtained
shows that the number observed (O) is in good fit with the number expected
(E).
MATERIALS:
Blue card
Green card
Plastic bag / beaker

Yellow card
Red card

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 until all 40 pairs of the cards are drawn.
5. Record all the combination in Table 1, and then count the X2 value.
Phenoty
pe
Tall
Short

Genoty
pe
TT
tt

(O-E)

(O-E)2

(O-E)2 / E

X2 =

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
Yellow cards
Green card
Red card

Allele
Allele
Allele
Allele

for
for
for
for

tall (T)
short (t)
purple flower (A)
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
Tall, purple
flower
Tall, white
flower
Short, purple
flower
Short, white
flower

Genoty
pe
TTAA

(O-E)

(O-E)2

(O-E)2 / E

TTaa
ttAA
Ttaa
X2 =

QUESTIONS:

1.

Do the monohybrid and dihybrid ratios obtained, fit the expected

Mendelian ratios?

2.

Compare your X2 value with the X2 from the class results. Which one
has lower X2 value? Explain.

PRACTICAL 4:KARYOTYPING
TITLE : KARYOTYPING
OBJECTIVE:
To determine gender and 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
colchicines 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
Handout

Glue
A4 paper

10

PROCEDURES:
1.

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

Chromosome 1
Chromosome 2

: The biggest chromosomes with centromere at the centre

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

2. The gender and abnormalities are determined from the arranged

chromosomes.
PRACTICAL 5
TITLE : MICROPIPETTING
OBJECTIVE:
To learn basic equipment in micropipetting.
INTRODUCTION:

11

Most reaction in DNA cloning and other manipulations of nucleic acids are
performed in small (0.2 1.5 ml) microcentrifuge tubes in volumes as small
as 10 l. Using such small reactions results in considerable savings in
amounts of reagents, enzymes and DNA, but you must be able to dispense
volumes as small as 0.5 l accurately. Micropipetters are capable of
dispensing such small volumes accurately, and thus it is essential that you
master micropipetting techniques in order to perform the experiments in this
manual properly. Various sized micropipetters, including those capable of
dispensing volumes up to 1000 l, will be used throughout the course, and it
is important to be able to use these correctly and precisely.
MATERIALS:
1. Beaker with sterile 1.5 ml microcentrifuge tubes (assorted colors: blue,
green, yellow and red)
2. Microcentrifuge tube opener
3. Microcentrifuge tube rack
4. Fine point permanent marker
5. Micropipettor stand
6. 0.5-10 l adjustable micropipettor
7. 10-100 l adjustable micropipettor
8. 100-1000 l adjustable micropipettor
9. Sterile 10 l micropipette tips
10.Sterile 100 l micropipette tips
11.Sterile 1000 l micropipette tips
12.Micropipette tip discard beakers
PROCEDURES:
1. Take four 1.5 ml microcentrifuge tubes.
2. With a permanent marker, label A D (for blue, green, yellow and red)
on the frosted lid and on the frosted labelling spot on the side.
3. Add volumes of the reagents to tube A D as indicates in the following
table.
Tube
Solution
A
475 l
B
256 l
C
300 l
D
25 l
4. Compare the volume with the standard microcentrifuge tubes and
carefully notice how much liquid is in the tip with each volume
pipetted. With practice you will be able to visually recognize if the
volume is accurate.
QUESTIONS:
1. Were you able to use the micropipettors accurately and gain familiarity
wuth what different volumes look like a tip?

12

2. What errors could result in pipetting volumes greater than desired

volume?
3. What errors could result in pipetting volumes smaller than desired
volume?
4. Micropipettors are not 100% accurate; accuracy and precision data are
generally provided by the supplier. For example, a typical 0.5 l - 10 l
micropipettor may be accurate to + 0.1 l. what is the percent error if
you are pipetting 10 l? What is the percent error if you are pipetting
0.5 l?

BASIC GENETICS LABORATORY PRACTICAL REPORT

For this course you will required to write up a report of the experiment that you
carried out for that week. Although there can be various in style, there are several
heading that should be common to all laboratory report. Following is a guide of what
is expected in a report for this subject.
......
Experiment Title : start each report by putting the title the tittle of the experiment at
the top of the page
Objective: maximum one or two sentences on exactly what you were hoping to
achieve during the experiment. Keep it short and clear.
Apparatus and material : separate the apparatus and the material used into two
different smaller portion. Bullet points can be used here.
Procedure : write down all the step that you have perform for thet particular
experiments in PASSIVE FORM and in a sentences NOT in point or bullet form.
Result : write down all raw data and observation. Data should be table if possible. Of
possible include one or two simple sentence to summaries what is your result at the
end of the section.
Discussion and question : discuss about the result your obtain. Tell the experiment
is success or unsuccessful. If the experiment is unsuccessful tell about the misconduct
and also the ways to prevent misconduct. Discuss about the theorical knowledge
about that experiment and compare with the practical. Answer the question based on
theorical and also practical.
Conclusion : tell about what is obtain from the experiments. Does your objective
achive.

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Fonts: times new roman 12, separate each section with BOLDED HEADING

RUBRIC REPORT
(To be attached together with the submission of lab report)

BEGINNING
(1 MARK)

Format

Report done
without
following the
given format.

DEVELOPING
(2 MARKS)

ACCOMPLISHE
D (3 MARKS)

EXEMPLARY
(4 MARKS)

Report done with

some error.

Report done by
following most of
the format.

Report done as
given format
accurately.

Content

Not organised
content.

Some content
start and end are
unclear. Not in
order.

Contents are
organised logically
but no diagram.

Good
organisation.
Contents are
logically,
ordered with
diagrams.

Originality
Informatio
n/
Plagiarism

Plagiarism,
copy and
paste from
Internet.

Details are
somewhat sketchy
and unable to find
specific details.

Some details are

non-supporting to
the subject.

Supporting
details specific
to subject with
coded reference.

SCOR
E

14

Neatness /
Creativity

Illegible
writing, loose
pages. No
illustration.

Legible writing,
some ill-formed
letters, print too
small or too large,
papers stapled
together. One
illustration.

Reference
& Time

No reference
coded. Report
handed in
more than one
week late.

Reference coded
does not support
the topic. Up to
one week late.

Legible writing,
some well-formed
characters, clean
and neatly bound in
a report cover.
Illustrations
provided.

Word processed
or typed, clean
and neatly bound
in a report cover.
Illustrations
provided
creatively.

One or two
reference coded. Up
to two days late.

References are
coded as given
format. Report
handed in on
time.
TOTAL

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