September, 2023

Laboratory
Manual GENETICS
This practical is designed to give
students an introduction to major
organisms used for studying
genetics. Student will have chance
to observe the steps in cell
division, to perform technique in
culturing fruit flies, and then Name:……………………………………..
distinguish male/female fruit flies,
to understand how the genetic ID:…………………………………………
material passed between
generations and how DNA can be
extracted from plant cells.

Prepared by Tong Thi Hang, MSc.

HCMC-2023
 CONTENTS

LABORATORY SAFETY GUIDELINES .......................................................2

LAB 1: Culturing and Experimental Mating of Drosophila melanogaster .......... 6

LAB 2: Mitosis.................................................................................................... 11

LAB 3: Meiosis ................................................................................................... 14

LAB 4: Extraction of DNA from plant cells ....................................................... 18

LAB 5: Plasmid extraction from E.coli culture …………………………… 21

LAB 6: Gel Electrophoresis ............................................................................... 23

APPENDIX - RECIPES .................................................................................... 25

HCMC-2023
School of Biotechnology – IU

LABORATORY SAFETY GUIDELINES

A. GENERAL LABORATORY SAFETY

1. Work carefully and cautiously in the laboratory, using common sense and
good judgment at all times.
2. EATING. DRINKING AND SMOKING ARE PROHIBITED in the
laboratory and in the laboratory space of a combined lecture/laboratory room.
3. Long hair must be tied back during laboratory sessions.
4. Open toed shoes are prohibited.
5. No sleeveless tops are permitted. Lab coats must be worn.
6. Identify the location of all exits from the laboratory and from the building.
7. Be familiar with the location and proper use of fire extinguishers, fire
blankets, first aid kits, spill response kits, and eye wash stations in the
laboratory.
8. Report all injuries, spills, breakage of glass or other items, unsafe conditions,
and accidents of any kind, no matter how minor, to the instructor immediately.
9. Keep sinks free of paper or any debris that could interfere with drainage.
10. Lab tables must be clear of all items that are not necessary for the lab
exercise.
11. Wash hands and the lab tables with the appropriate cleaning agents before
and after every laboratory session.

B. OPEN FLAMES - FIRE HAZARD

1. Identify and be familiar with the use of dry chemical fire extinguishers that
are located in the hallways and laboratory rooms.
2. Flames are only to be used under the supervision of the instructor.

C. SHARP OBJECTS AND BROKEN GLASS

1. Pointed dissection probes, scalpels, razor blades, scissors, and microtome
knives must be used with great care, and placed in a safe position when not in
use.

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2. Containers designated for the disposal of sharps (scalpel blades, razor

blades, needles; dissection pins, etc.) and containers designated for broken
glass are present in the laboratory. Never dispose of any sharp object in the
regular trash containers.
3. Report all cuts, no matter how minor, to the instructor.
4. All biology labs and the biology preparation room (702) house a first aid kit
containing antiseptics, bandages, Band-Aids and gloves to care for minor cuts.
5. Do not touch broken glass with bare hands. Put on gloves and use a broom
and dustpan to clean up glass. Dispose of ALL broken glass in the specific
container marked for glass. Do not place broken glass in the regular trash.
6. When cutting with a scalpel or other sharp instrument, forceps may be used
to help hold the specimen. Never use fingers to hold a part of the specimen
while cutting.
7. Scalpels and other sharp instruments are only to be used to make cuts in the
specimen, never as a probe or a pointer.

D. INSTRUMENTS AND EQUIPMENT

Care must be used when handling any equipment in the laboratory. Students
are responsible for being familiar with and following correct safety practices
for all instruments and equipment used in the laboratory.

Microscope Handling
1. Microscopes must be carried upright, with one hand supporting the arm of
the microscope and the other hand supporting the base. Nothing else should be
carried at the same time.
2. Microscope must be positioned safely on the table, NOT near the edge.
3. After plugging the microscope into the electrical outlet, the cord should be
draped carefully up onto the table and never allowed to dangle dangerously to
the floor.
4. The coarse adjustment must NEVER be used to focus a specimen when the
40x or oil immersion lens is in place.
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5. When finished with the microscope, the cord should be carefully wrapped
around the microscope before returning it to instructor.
6. The microscope must be placed upright and in the table near the cabinet.
7. All prepared microscope glass slides are to be returned to their appropriate
slide trays; wet mount preparations are to be disposed of properly.
8. Malfunctioning microscopes should be reported to the instructor.

Hot Plates and Water Baths

1. The instructor will regulate the temperature of hot plates and water baths
with a thermometer.
2. This equipment must be placed in a safe place.
3. Use insulated gloves or tongs to move beakers or test tubes in and out of the
water baths.
4. Use care when working near hot plates and water baths, as they may still be
hot even after being turned off.

E. ADDITIONAL LABORATORY SAFETY FOR THE GENETICS LAB

1. The Drosophila melanogaster that are used for the lab experiments are not fit
for the natural environment and are mutations of the wild form of this animal.
They will not be released into the open air but euthanized in mineral oil.
2. Fly nap (triethylamine and ethanol) is to be used only as directed by the
instructor and following the manufacturers recommendations.
3. DNA recombination experiments will only include DNA from the bacteria
E. coli and it's plasmids. These recombinant organisms will be disposed of in
biohazard receptacles.
4. E. coli strains used in the laboratory are not pathogenic and cannot survive in
the adult large intestine.
5. Protective goggles will be used when viewing gels illuminated with
ultraviolet light.
6. Gloves will be worn during molecular experiments and will be changed if
there is a tear or spill.
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7. When agarose gels are stained with ethidium bromide only the instructor will
handle the gel and the staining solution. Ethidium bromide is a mutagen by the
Ames test and suspected carcinogen.

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LAB 1: CULTURING AND EXPERIMENTAL MATING OF

DROSOPHILA MELANOGASTER

I. Introduction
Drosophila melanogaster is a cosmopolitan species, which can be found
all over the world, including in your home if you have overripe fruits in the
summer. Fruit fly has been widely used for genetic investigation because it has
many characteristics of a good experimental model, including the short life
cycle, the ease of culturing, the high reproductive rate, and the small
chromosome number.

General structure of the adult fruit fly

Drosophila’s body has three main parts: the head, the thorax, and the
abdomen. The major structures of the head are two big compound eyes, two
antennas, a mouth. The thorax has six legs, two wings, and two halteres, which
are small, club-shaped structures behind the wings that ensuring the fly balance
when flying. On the dorsal surface of the thorax, there are a number of long
dark bristles.
It is not difficult to distinguish a female from a male fly. The male is
usually smaller than the female. In addition, the female has more pointed
abdomen, which is stripped rather than back-tipped (Fig. 1.1). Other features
that can be used to determine the sex of the adult D. melanogaster is shown in
Table 1.1.
Table 1.1 Features for sex determination of fruit fly.

Characteristics Male Female

Overall size Generally smaller the female Generally larger than

male
Size of abdomen Smaller than female’s Larger – due to
distention with
maturing eggs
Shape of abdomen Narrower than female Broader

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Tip rounded (blunt) Tip is pointed

Color of tip of Solid black dorsally Not so

abdomen

Penis Small, dark spot on ventral tip of Absent

abdomen. This, with dorsal black area,
makes tip of abdomen appear entirely
black

Number of 5 7
abdominal
segments visible

Sex comb# Present; about 10 dark, short, curved Absent

bristles (tufts) on the front legs
#
Sex comb is special bristle found on the front legs of the male fruit fly. It has no sexual
function and can be observed at the 100-fold magnification.

Life cycle
Drosophila life cycle consists of the following four main stages (Fig. 1.1)
• Eggs. Eggs are laid by the mother on the food and take about one day to
hatch. They are small and translucent with two “ears” sticking out.
• Larva. Larvae are maggots, which crawl through the food in jerky motion,
eating as they go. The larvae go through three molts: they are hatched
from the egg as small, “the first instar larvae”. Then after a day they molt
to become larger, “the second instar larvae”. After two days, “the third
instar-larvae” climb up the wall of the vial, glue themselves to the glass,
invert their spiracles (breathing tubes), and settle down as pupae.
• Pupa. Pupae are the cocoons in which the larvae metamorphose into
adults. The larval cuticle becomes a shell, its muscle melt away, and the
new adult exoskeleton as well as musculature form inside. The pupal
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stage lasts five days. In the last day, you can see the red eyes and the dark
wings forming inside.
• Adult. The adult emerges from the pupal case as a white, elongated fly,
whose wings are stilled folded up. After about an hour, the wings will
expand and the body will take its normal shape and coloration. The adults
become sexually mature after 8-10 hours. After this time, the males chase
the females about in an endless quest for mating. Flies can live up to three
months, but they are pretty decrepit after 6 weeks or so.

Figure 1.1 Life cycle of Drosophila melanogaster

The length of life cycle and each stage vary according to the temperature.
Average lengths of developmental periods are as follows.

Temperature 10oC 15oC 20oC 25oC 28oC 30oC 31oC

Egg-Larval 57.0 17.3 8.1 5.0 4.2 4.0 5.0
Pupae-Adult 13.7 13.3 6.5 4.2 3.4 3.4 3.4
Total (days) 70.7 30.6 14.6 9.2 7.6 7.4 8.4

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II. Objectives
Upon completion of this investigation, the student should be able to
• Culture fruit flies in vials,
• Distinguish male and female fruit flies, and
• Identify various stages in the life cycle of D. Melanogaster.

III. Materials
Saccharose Forceps
Propionic acid Small pain brushs
Dry baking yeast Petri dish
Agar Etherizer flask
Water Funnel
Ether Cotton mesh
Culture tube Sterile tissue papers
250 ml beaker Stereo dissecting microscope

IV. Procedure
A. Preparing media
Each group of students will prepare 4 medium vials for culturing of the
fruit fly
1. Boil 0.75 g agar in 50 ml of water until the agar completely dissolve
2. Add 2.5 g of saccharose and 2.5 g of dry baking yeast, and then boil the
mixture for 1 minute with occasional stirring.
3. Add 250 µl of propionic acid and stir well.
4. Pour the medium into the sterile vials to the depth of about 2 cm. Take
care to prevent the medium from coming into contact with the neck of the
vial.
5. Close the vial of the cotton plug, and leave the medium to cool to room
temperature.
6. Before put the flies into the tubes for culturing, dry the inner wall of the
tube with sterile tissue papers.
B. Handling flies

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1. Put some drops of ether on a piece of cotton mesh inside the funnel that is
put on top of the etherizer flask.
2. Strike the base of the culture tube lightly on the palm of the hand so the
flies will drop to the bottom.
3. Remove the plug and quickly invert the tube over the funnel.
4. Expose the flies to ether for about 1-2 minutes after they stop moving.
Overetherization will kill the flies.
5. Transfer some etherized flies to a dry petri dish.
6. Examine the flies under the dissecting microscope with the light source
shining from above. Use a small paintbrush to move the flies about on the
petri dish.
7. Divide the flies in to two groups based on their sex.
8. Open the plug of the culture tube, keep the tube horizontally and gently
transfer three female and three male flies into the tube.
9. Wait until the flies revive before turn the tube vertically otherwise the
flies come into contact with the moist medium, wetting their wings and
unable to fly out of the medium.
10. Keep the tubes in cool place for one week or more.
11. Observe the tube to identify different stages in the life cycle of
Drosophila. The third instar larvae will be used for practical 6.

V. Report
1. Record the following morphological characteristic of flies that you
observe:
• Body color: ..........................................................
• Eye color: ............................................................
• Wing shape:.........................................................
• Presence of antenna:............................................
2. Record morphological states of the Drosophila that you can observe in
the tube after one and two weeks.
3. Record the different characteristics of male and female fruit flies

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LAB 2: MITOSIS

I. Introduction
Mitotic cell division is the type of cell division that results in the
formation of two genetically identical daughter cells from a single mother cell.
Before entering mitotic cell division, cell spends long time in the interphase to
acquire or synthesize materials that are essential for cell division and to
replicate its DNA. Mitotic cell division consists of two consecutive processes:
mitosis (nuclear division) and cytokinesis (cytoplasmic division). During
mitosis, duplicated chromosomes, each consists of two sister chromatids,
condense into visible threadlike structures then equally separated into the
opposite end of the cell. Following mitosis, nucleus is formed in each daughter
cell and the cytoplasm is divided into each daughter cells through the process
of cytokinesis.
Mitosis can be divided into four phases: prophase, metaphase, anaphase
and telophase (Fig. 2.1):
During prophase, the chromosomes condense and the spindle microtubles
form and attach to the chromosomes. The site on chromosome that binds to the
spindle is called kinetochore. Kinetochores of sister chromatids are tethered to
the spindles that are originated from opposite pole of the cell. Nuclear envelope
breaks down and the nucleolus disappears.
During metaphase, the chromosomes line up in a single plane (the
metaphase plate) along the equator of the cell.
During anaphase, chromatids of each chromosome separate and move to
the opposite poles of the cell. The chromatids form V-liked structures with the
centromeres pointing toward the respective poles.
Telophase starts when the chromosomes reach the poles. During this
phase, the chromosomes relax, the nuclear envelope is re-synthesized around
both groups of chromosomes, the spindle apparatus is dismantled, and the
nucleoli reappear. Cytokinesis usually occur during telophase, separating the
two nuclei into separate cells.
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Interphase

Prophase

Metaphase

Anaphase

Telophase

Figure 2.1 Chromosomes’ behavior during mitotic cell division

II. Objectives
Upon completion of this investigation, the student should be able to

• Outline the procedure to prepare acetocarmin squashes of onion root tips,

and
• Describe in chronological order the main events of mitosis in onion root
tip.

III. Materials
Onion bulbs Microscope
1M HCl Microscope slides & coverslips
Distilled water Watch glasses
70% & 90% ethanol Forceps
Acetocarmin stain Scissors
Carnoy’s solution Sand

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IV. Procedure
Before the practical, your instructor put the onion bulbs on top on moisten
sand until their roots grow to about 5 cm in length. Approximately 1 cm of the root
tip was collected and soaked in Carnoy’s solution for 12 h. Next the roots were
washed twice with 90% ethanol for 10 minutes each time. They were then stored in
70% ethanol until use.
Each student will prepare acetocarmin-stained squash of onion roots by the
following procedure:
1. Use a forceps to transfer some onion roots into a watch glass.
2. Wash the roots with water 2-3 times to remove the ethanol.
3. Remove all water then soak the roots in several drops of 1M HCl for 10-15
minutes to soften them. After that, wash the roots 2-3 times with water.
4. Soak the roots in acetocarmine stain for 15-20 minutes.
5. Put one drop of water at the middle of a microscopic slide then transfer one
stained onion root into the water drop.
6. Cover the root with a coverslip. Avoid creating bubble under the coverslip.
7. Gently apply pressure with your thumb over the cover glass to squash the root
tip into a thin layer.
8. Examine the specimen under low power (10X objective) to identify the
meristem area, where dividing cells are located. Then use higher power
objective lens to carefully examine cells in various mitotic stages.

V. Report
Draw and clearly label cells in various mitotic phases that you observe.

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LAB 3: MEIOSIS
I. Introduction
Gametes, cells specialized for sexual reproduction, are haploid, carrying only a
single set of chromosomes and thus, only a single copy of the organism genetic
information. Gametes are formed by the process of cell division called “meiosis”.
This division process results in the reduction of chromosome number from diploid
in the mother cells to haploid in the daughter cells. This reduction in chromosome
number is essential because when two gametes fuse in fertilization, the
chromosome number is restored to diploid in the embryo. Like mitotic process,
meiosis is preceded by an interphase, when genomic DNA of the cell is replicate,
doubling the number of chromosomes. The interphase is followed by two rounds of
cell divisions, namely meiosis I and meiosis II. Each of these divisions is divided
into prophase, metaphase, anaphase, and telophase, which can be differentiated by
the roman number following the name of each phase (Fig. 4.1). The period between
meiosis I and meiosis II is “interkinesis”, in which the nuclear membrane re-forms
around the chromosomes clustered at each pole, the spindle breaks down, and the
chromosomes relax. The behavior of chromosome during meiosis (Fig. 4.1) is
summarized as follows:
Prophase I; Prophase I is a lengthy stage, in which chromosomes contract and
become visible; homologous chromosomes begin to pair up forming tetrads or
bivalents; and crossing over (synapsis) take places. Near the end of prophase I, the
nuclear membrane breaks down and the spindle forms.
Metaphase I: The tetrads move toward the center and line up on the metaphase
plate. As tetrads align themselves in the middle of the cell, they attach to spindle
fibers in a unique manner: the centromeres of homologous chromosomes attach to
separate spindles, each from different poles. Nuclear envelope completely
disappears.
Anaphase I: The unique even occurring at this phase is the separation of the
homologs. In contrast to mitotic anaphase, the centromeres of a given chromosome
do not divide. As a consequence, chromosomes of each homologous pair move
toward opposite poles, resulting in the halving of chromosome number in the
daughter cells.

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Telophase I: The chromosomes arrive at the spindle poles and cytoplasm divides.
The chromosomes, however, do not completely uncoil.
Prophase II: The chromosomes recondense; the spindle re-forms; and nuclear
envelope once again breaks down.
Metaphase II: Individual chromosomes line up on the metaphase plate with sister
chromatids facing the opposite poles.
Anaphase II: The kinetocores of sister chromatids separate and the chromatids are
pulled to the opposite poles. Each chromatid is now a distinct chromosome.
Telophase II: The chromosomes arrive at the spindle poles; nuclear envelope re-
forms around the chromosomes; and cytoplasm divides. The chromosomes relax
and are no longer visible.

Figure 4.1 Meiosis process

II. Objective
Upon completion of this investigation, the student should be able to
• Describe the main events of meiosis,
• Identify and draw cells in garlic chive flower at different meiotic phases, and

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• Compare and contrast between meiosis and mitosis.

III. Material
Garlic chive flower Microscope
70 % Ethanol & 90% Ethanol Microscope slide & coverslip
Carnoy’s solution Dissecting needle
Aceto-orcein stain Forceps
1N HCl Immersion oil
10% glycerol

IV. Procedure
Before the practical, the instructor collected garlic chive flowers and soaked
them in carnoy’s solution for 12-20 hours. The flowers were then washed twice
with 90% ethanol (10 minutes each time) and stored in 70% ethanol.
1. Each students use a forceps to transfer some garlic chive flowers into a
watch glass, and use dissecting needles to remove the perianth and petals
from the flowers, only retain the anthers.
2. Wash the anthers with water 3 times.
3. Completely remove water from the watch glass. Be careful, not pour the
tiny anthers down the sink.
4. Add several drop of 1N HCl solution into the watch glass and soak the
anthers in this solution for 10 minutes.
5. Wash the anthers with water 3 times.
6. Completely remove water, then add several drops of aceto-orcein stain
into the watch glass.
7. Soak the anthers in staining solution for 30 minutes.
8. Transfer four stained anthers to a microscope slide, which is pre-mounted
with one drop of 10% glycerol. Cover with a coverslip.
9. Use the handle of dissecting needle to gently apply pressure over the
coverslip. This should squash the anthers into a thin cell layer.
10. Observe the specimen under low power objective to identify the area with
many dividing cells. Then use higher power objective lens to carefully
observe cells with different meiotic phases.

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V. Report
1. Draw and clearly label cells at various stages of meiosis that you can
observe.
2. Compare the similarities and differences between the mitosis and the
meiosis.

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LAB 4: EXTRACTION OF DNA FROM PLANT CELLS

I. Introduction
DNA is genetic material of the cells and organisms. All genetic information of
cells or organisms is included in DNA.
DNA extraction from plant tissue can vary depending on the material used.
Essentially any mechanical means of breaking down the cell wall and
membranes to allow access to nuclear material, without its degradation is
required. For this, usually an initial grinding stage with liquid nitrogen is
employed to break down cell wall material and allow access to DNA while
harmful cellular enzymes and chemicals remain inactivated. Once the tissue
has been sufficiently ground, it can then be resuspended in a suitable buffer,
such as SDS. In order to purify DNA, cell debris then will be precipitated by
centrifugation. DNA must then be precipitated from the aqueous phase and
washed thoroughly to remove contaminating salts. The purified DNA is then
resuspended and stored in TE buffer or sterile distilled water. This method has
been shown to give intact genomic DNA from plant tissue. To check the
quality of the extracted DNA, a sample is stained with ethidium bromide, and
visualised under UV light.

II. Objectives
Upon completion of this investigation, the student should be able to
- Extract DNA from plant cells
- Recognize the presence of DNA in the extraction.

III. Tools and Materials:

200mg plant tissue
Eppendorf
Mortar and Pestle
Microcentrifuge
Absolute Ethanol (ice cold)
70 % Ethanol (ice cold)

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3 M Sodium Acetate, pH5.2

550 C water bath
Water (sterile)
GelRed loading buffer
SDS buffer

IV. Procedure
1. Grind 200 mg of plant tissue to a fine paste in approximately 500μl of
SDS buffer.
2. Transfer SDS/plant extract mixture to an eppendorf.
3. Incubate the SDS/plant extract mixture for about 15 min at 550C in a
recirculating water bath.
4. After incubation, spin the SDS/plant extract mixture at 12000 g for 5
min to spin down cell debris. Transfer the supernatant to a clean
eppendorf.
5. Add 1 volume of Chloroform: Iso Amyl Alcohol (24:1) to 1 volume of
supernatant and mix the solution by inversion.
6. After mixing, spin the tubes at 13000 rpm for 1 min.
7. Transfer the upper aqueous phase only (contains the DNA) to clean
eppendorfs. Repeat this step if neccessary
8. To each eppendorf add 0.1 volume of sodium acetate 3M pH 5.2 and 2.5
volumes of ice cold absolute ethanol.
9. Invert the tubes slowly for a few times to precipitate the DNA.
Generally the DNA can be seen to precipitate out of solution.
10. The tubes can be placed for 30 minutes to 1 hr at -200C after the
addition of ethanol to precipitate the DNA.
11. Spinning the tube at 13000 rpm for 3 minutes to form a pellet.
12. Remove the supernatant and wash the DNA pellet by adding 500μl ice
cold 70 % ethanol and spinning the tube at 13000 rpm for 3 minute.
13. Remove the supernatant. Wash the precipitate again with ice cold 70%
ethanol.

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14. After the wash, Remove all the supernatant and allow the DNA pellet to
air dry (approximately 15 min). Do not allow the DNA to over dry or it
will be hard to re-dissolve.
15. Resuspend the DNA in sterile DNase free water (approximately 50-
400μl H2O; the amount of water needed to dissolve the DNA can vary,
depending on how much is isolated).
- RNaseA (10 μg/ml) can be added to the water prior to
dissolving the DNA to remove any RNA in the preparation (10 μl
RNaseA in 10ml H2O).
- After resuspension, the DNA is incubated at 650C for 20 min to
destroy any Dnases that may be present and store at 40C.

17. Check the presence of DNA in the solution using spectrometer or

electrophoresis.

V. Report.
1. Introduction to the work
2. Materials and methods
3. Results
4. Discussions
i. Explain the function of SDS buffer?
ii. Function of sodium acetate?
iii. What is the color of DNA after extration?
iv. How do you know you extraction contain DNA?
5. Conclusion
6. References

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LAB 5 : PLASMID EXTRACTION FROM E.COLI CLONE

I-INTRODUCTION
Plasmid is small circular DNA molecule, present in many microbial cells with
common size about 2 up to 500 kb. These molecules are mobile factors which
carry several genes for the survival and fitness of organisms. Plasmids could be
transferred from cells to cells, so that it has been utilized as vector to carry
genes for studying the gene expression and genetic engineering. Preparation of
plasmid includes extraction, construction, and maintenance.
In this lab section, the BlueScript plasmid was transformed and maintained in
E.coli cloning strain for later manipulation. The in-lab practice requires
students to culture bacterial cells and extract plasmid. Extracted plasmid is
checked in agarose gel electrophoresis.

II- OBJECTIVES
Upon completion of the work, student can:
- Collect E.coli cells for plasmid
- Open circular plasmid with restriction enzyme
- Distinguish plasmid extraction and genomic DNA extraction.

III- PROCEDURE
III.1 Plasmid extraction
1. Grow 2 mL overnight cultures from single colonies of bacteria containing
your plasmid of interest.
2. Add 1.5 mL of the stock culture to a 1.75 mL microfuge tube.
3. Centrifuge in microfuge tube at 10,000 g for 30 sec.
4. Pour off the supernatant, being careful not to disturb the bacterial pellet.
5. Resuspend the pellet in 100 μL of cold Solution I.
6. Vortex the solution for 2 min or until all bacteria are fully resuspended.
Add 10mg lysozyme to the mixture.
7. Add 200 μL of Solution II and invert the tube carefully 5 times to mix the
contents. The contents will become clear and thicker as the proteins and
DNA are denatured (Do not vortex at this stage or the genomic DNA will

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become sheared and will therefore contaminate your purified plasmid

DNA.)
8. Incubate solution on ice for 5 min.
9. Add 150 μL of cold Solution III to each tube.
10. Mix by inverting several times. A white precipitate will be formed which
contains the bacterial proteins and genomic DNA.
11. Incubate tube on ice for 5 min.
12. Centrifuge the tube for 5 min at 12,000 g.
13. Collect the supernatant into a new tube by pipetting or carefully pouring.
14. (Optional) Add 5 μL of 2 mg/mL RNase A to the supernatant in the new
tube and incubate at 37 °C for 5 min.
15. (Optional) Perform phenol-chloroform extraction
16. Add either 700 μL of cold 100% ethanol or 350 μL room temperature
isopropanol to the solution to precipitate the plasmid DNA for 20 minutes
17. Pour out the supernatant.
18. (Optional) Wash the pellet with 70% ethanol.
19. Air dry the pellet (can be done by inverting the tube at an angle over tissue
paper) for 20-30 min.
20. Resuspend pellet with 25-50 μL of TE.
III.2. Digestion of plasmid with restriction enzyme
To check plasmid in agarose gel electrophoresis, extracted circular plasmid is
opened with restriction enzyme. Using enzyme with 1 cutting site in plasmid
(such as BamH1), plasmid is linearized. Following the instruction from the
manufacturer (Promega), the recommended reaction condition is:
Components Volume
Plasmid DNA 1 µg/µl 1 µl
RE buffer 10X 2 µl
Acetylated BSA, 10 µg/µl (optional, for fast digest in 15 minute) 0.2 µl
H2 O 15.8 µl
Mix by pipetting then add:
RE BamH1 1* µl
Final volume 20 µl
*The number of restriction enzyme units added to the reaction varies at 5–12 units, depending on the concentration
of restriction enzyme used.

Mix gently by pipetting, close the tube and centrifuge for a few seconds at in a
microcentrifuge. Incubate at 37oC for 45 minutes – 1 hr.

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LAB 6: DNA QUALITY AND QUATIFICATION

I. Background
Gel electrophoresis is the standard lab procedure for separating DNA by size
(e.g. length in base pairs) for visualization and purification. Electrophoresis
uses an electrical field to move the negatively charged DNA toward a positive
electrode through an agarose gel matrix. The gel matrix allows shorter DNA
fragments to migrate more quickly than larger ones. Thus, the length of a DNA
segment can be accurately determined by running it on an agarose gel
alongside a DNA ladder (a collection of DNA fragments of known lengths).
II. Materials and method
Materials
The equipment and supplies necessary for conducting agarose gel
electrophoresis are relatively simple and include:
- An electrophoresis chamber and power supply.
- Sample combs, around which molten agarose is poured to form sample
wells in the gel.
- Electrophoresis buffer, usually Tris-acetate-EDTA (TAE), Tris-borate-
EDTA (TBE) or Sodium Boric acid (SB) buffers.
- Loading dye, which contains something dense (e.g. glycerol) to allow
the sample to "fall" into the sample wells, and one or two tracking dyes,
which migrate in the gel and allow visual monitoring or how far the
electrophoresis has proceeded.
- Ethidium Bromide, a fluorescent dye used for staining nucleic acids.
- Transilluminator (an ultraviolet light box), which is used to visualize
EtBr-stained DNA in gels.
- DNA 100 bps ladder
- Agarose
Method
1. Place the gel plate into gel mould, position the comb
2. Prepare 1.5% agarose gel: dissolve 1.5 grams of agarose and add to the
100ml 1XSB buffer solution. Heat the mixture in a microwave oven until
completely dissolved. Let the solution cool at room temperature to 60oC.
It is a good idea to microwave for 30-45sec, stop and swirl, and then continue towards a boil.
Keep an eye on it as the initial boil has a tendency to boil over.

3. Add 5 µl of EtBr into the solution

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Caution EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when
working with this chemical.

4. Pour the agarose into a gel tray with the well comb in place, and allow it to
set for at least 30 minutes.
Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away
from the well comb or towards the sides/edges of the gel with a pipette tip.

5. Remove the comb. Place the gel into the gel electrophoresis chamber and fill
gel box with 1x SB buffer until the gel is covered.

Loading Samples and Running an Agarose Gel

1. Add loading buffer to each of your samples.
2. Carefully load 5µl of DNA ladder into the first well of the gel.
3. Run the gel at 80-150V until the dye line is approximately 75-80% of the
way down the gel.
4. Turn OFF power, disconnects the electrodes from the power source, and then
carefully removes the gel from the gel box.
5. Using any device that has UV light, visualize your DNA fragments.
6. Using UV Transilluminator to visualize your DNA fragments
(CAUTION: UV LIGHT IS HAZARDOUS!!! PLEASE WEAR MASK OR
UV PROTECTION GLASSES IF EXPOSED TO UV LIGHT).
7. Switch on the Transilluminator. Photograph the gel under UV light.

Analyzing Your Gel:

Using the DNA ladder in the first lane as a guide (the manufacturer's
instruction will tell you the size of each band), you can interpret the bands that
you get in your sample lanes to determine if the resulting DNA bands that you
see are as expected or not.
Quality of extracted DNA can be assessed by looking at gel. Good quality
DNA will show as a sharp intense band. Degraded DNA extracts will show
various degree of smearing

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APPENDIX – RECIPES
Reagent composition
Carnoy’s solution:
- Mix absolute ethanol and glacial acetic acid with the following ratio:
3 vol. of ethanol: 1 vol. of acetic acid
- Prepare fresh.
Acetocarmin
Carmin 0.1g
45% Glacial acetic acid 10ml
10% Ferric chloride (FeCl2*6H2O) 0.5ml
- Boil 10 ml of 45% glacial acetic acid
- Add 0.1 g of carmin.
- Add 0.5 ml of 10% Ferric chloride (this step is optional).
- Cool rapidly.
- Filter and store in the dark.
Aceto-orcein
Orcein 0.1g
Glacial acetic acid 5.5ml
ddH2O ~ 4.5ml
- Boil 5.5ml of glacial acetic acid then add 0.1g of orcein powder.
- Cool the solution.
- Add distilled H2O to 10 ml and filter.
- Store in the dark.
SDS buffer
- Tris-HCl 1M, pH7.4 10ml
- EDTA 0.5M, pH8 2.5ml
- NaCl 5M 2.5ml
-SDS 10% 2.5ml
- ddH2O 32.5ml

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Solution I - Resuspension Buffer

1. 25 mM Tris-HCl (pH 8)
2. 50 mM glucose
3. 10 mM EDTA

Store Solution I at 4°C

Solution II - Denaturing Solution

4. 0.2 N NaOH
5. 1.0% SDS

Store Solution II at room temperature

Solution III - Renaturing Solution (Potassium Acetate)

6. 120 mL 5M Potassium acetate

7. 23 mL glacial acetic acid
8. 57 mL of dH2O

Store Solution III at 4°C

References
1. Genetics Laboratory Manual. Second edition. 2000. University of South Florida,
Tampa. James R. Garey, Samantha R. Brown, Laurie L. Markham, Richard A.
Anthony
2. TCU genetics lab manual. First edition. 2003. Texas Christian University. Phil
Hartman and Misti Caudle.
3. Laboratory manual for bios 308. Genetics. Dept. of Biological Sciences. Northern
Illinois University. DeKalb IL 60115. USA. Mitrick A. Johns.

Giảng viên phụ trách Trưởng bộ môn Trưởng Khoa

Tong Thi Hang

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