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Ministry of Higher Education

King Abdul Aziz University


Faculty of Science
Department of Biological science

Laboratory Manual of

Plant Biotechnology
Part 1
Dr. Jehan Salem
I. Bayan Sajer

1
Page# Safety and regulation General information about
1 PP lecture
3 safty
Physical and
Chemical sterilization A display of the sterilization
2 P#6 PP lecture
methods we have in the lab

Components of plant
PP lecture
culture medium Lab hand Making medium for plant
3 P #10
out tissue culture
Medium Preparation practical
Micro
Educational Explain how to grow in vetro
4 P #14 propagation video plants
Micro Lab hand
5 propagation cont. out Grow in vetro plants
practical
6 VACATION

The Difference of the


Genomic DNA
7 P #18 Extraction Between PP lecture
Animal & Plant

DNA extraction from a


Lab hand
plant General way How to extract plant DNA by
8 P #20 out
(detergent) detergents
practical

DNA extraction from a Lab hand


How to extract plant DNA by
9 P#24 leaf with chemicals out
chemicals
practical
Running plant DNA Lab hand
extracted by different out
10 P # 25 How to use electrophoresis
methods in Agarose practical
gel and
Detection of
Genetically Modified
11 P# 35 Foods by PCR PP lecture

2
The Final Exam
12
Lab 1
Safety and regulation

What is safety?
Elimination of potential threats to human health

Hazards and risk assessment:

- Hazard means the equipment, chemicals and conditions that have a potential to cause
harm such as chemicals ,electricity, animals and infectious agent .

- Risk is the probability that a hazard will cause harm.

- Risk assessment is an attempt to estimate the potential for human injury or property
damage from an activity.

- Safety guideline and standards are procedures that are designed to prevent
accidents by reducing the risk of hazards in situation where the hazards cannot be
eliminated entirely.

Laboratory Safety Management


1- Regulatory agencies

Regulations and Standards.


Categories of Regulations and Standards.
- Worker safety

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- Environmental protection
- The use and handling animals
- Regulation of radioisotopes.

2- Institutional Responsibility.

- Worker safety regulation

- Environmental Protection

Laboratory Responsibility:

- Risk reduction
-Labeling and Documentation
Material Safety Data Sheet (MSDS)
Job Safety analysis (JSA)
Housekeeping
Emergency response

PERSONAL RESPONSIBILITY:
• Be sure that you are informed about the hazard in the laboratory.

• When in doubt about hazards material or procedure ask.

• Use a personal protective wear such as lab coat and free powdered gloves all the
times.

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• Do not eat, drink or chew in the laboratory.

• Avoid practical jokes or horseplay.

• Wash your hands regularly before leaving the laboratory.

• Read the labels of chemicals carefully .

• Read procedures before performing them and visualize hazards steps.

• Minimize use of sharp objects and be sure that you properly dispose of them.

• Clean up any spills and pickup any dropped items.

• Label every thing clearly.

• Use fume hood for any chemicals or solvent that you can smell.

• Record every thing in your lab notebook.

• Always report accidents .

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Lab 2

Physical and Chemical sterilization

In most laboratory settings, the control of microorganisms is of paramount concern.

Decontamination is : the destruction or removal of microorganisms from instruments,


materials, body surfaces, etc.

Many agents and procedures have been developed to accomplish this end.
A. Generally, decontamination involves physical and/or chemical agents.

Physical agents include:

high temperature, radiation, filtration or sound waves.

Chemical decontamination agents are substances that react with and thus alter some
important molecular component of the cell.

Microorganisms are not uniformly affected by physical and chemical decontamination.


Susceptibility to the effects of physical and chemical agents depends upon the type of
microorganism and at what stage in the microorganism’s lifecycle they are exposed to
the agent. When choosing and applying a method of decontaminating materials, it is
important that you understand what type of organism is being targeted and the relative
resistance of that organism.
1. The target with the highest resistance is the bacterial endospores. Endospores are
ubiquitous in the environment.
2. Targets with the moderate resistance include cysts, sexual fungal spores,
nonenveloped viruses , Mycobacterium tuberculosis, Staphylococcus aureus and
members of the genus Pseudomonas.
3. Targets with the least resistance include vegetative cells of most microbes,
enveloped viruses , and asexual fungal spores.

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Definitions :
1. Sterilization refers to any process that destroys or removes all infectious organisms
including endospores and viruses.
2. Disinfection refers to any physical process or application of any chemical that will
kill the growing (vegetative) microbial cells. These processes need not kill or inactivate
endospores. A disinfectant is a chemical capable of killing microbial cells. It should be
understood that if a chemical is referred to as a disinfectant, it is to be used on
inanimate objects and not to be used on body surfaces.

3. Sanitize refers to any mechanical process (scrubbing, rinsing, etc.) that reduces the
microbial load on a surface. Sanitizers are chemical agents that assist in this task.
These are usually soaps or detergents.

4. Microbicidal agents are chemicals that will kill or destroy microorganisms. Among
the microbicidal agents are those that target specific microorganisms including:
a. fungicidal agents which are designed to kill fungi;
b. bactericidal agents which are designed to kill bacteria;
c. sporicidal agents which are designed to destroy endospores;
d. viricidal agents which are designed to destroy viruses.

5. Microbiostasis refers to the inhibition of growth of microorganisms. This does not


mean that the organisms are killed simply that they are unable to grow. Refrigeration
and many antimicrobial drugs exert a microbistatic effect.
a. Bacteriostatic agents are chemicals that inhibit the growth of bacteria.
b. Fungistatic agents are chemicals that inhibit growth of fungi.

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Sterilization techniques:

WET HEAT (Autoclaving)

The method of choice for sterilization in most labs is autoclaving; using pressurized
steam to heat the material to be sterilized. This is a very effective method that kills all
microbes, spores and viruses.

Autoclaving kills microbes by hydrolysis and coagulation of cellular proteins, which is


efficiently achieved by intense heat in the presence of water.

The intense heat comes from the steam. Pressurized steam has a high latent heat; at
100degC it holds 7 times more heat than water at the same temperature. This heat is
liberated upon contact with the cooler surface of the material to be sterilized, allowing
rapid delivery of heat and good penetration of dense materials.

At these temperatures, water does a great job of hydrolysing proteins… so those bugs
don’t stand a chance.

DRY HEAT (Flaming, baking)

Dry heating has one crucial difference from autoclaving. You’ve guessed it – there’s no
water, so protein hydrolysis can’t take place.

Instead, dry heat tends to kill microbes by oxidation of cellular components. This
requires more energy than protein hydrolysis so higher temperatures are required for
efficient sterilization by dry heat.

For example sterilization can normally be achieved in 15 minutes by autoclaving at


121degC, whereas dry heating would generally need a temperature of 160degC to
sterilize in a similar amount of time.

FILTRATION

Filtration is a great way of quickly sterilizing solutions without heating. Filters, of course,
work by passing the solution through a filter with a pore diameter that is too small for
microbes to pass through.

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Filters can be glass funnels made from heat-fused glass particles or, membrane filters
made from cellulose esters. For removal of bacteria, filters with an average pore
diameter of 0.2um is normally used.

But remember, viruses and phage can pass through these filters so filtration is not a
good option if these are a concern

SOLVENTS

Ethanol is commonly used as a disinfectant, although since isopropanol is a better


solvent for fat it is probably a better option.

Both work by denaturing proteins through a process that requires water, so they must
be diluted to 60-90% in water to be effective.

Again, it’s important to remember that although ethanol and IPA are good at killing
microbial cells, they have no effect on spores.

RADIATION

UV, x-rays and gamma rays are all types of electromagnetic radiation that have
profoundly damaging effects on DNA, so make excellent tools for sterilization.

The main difference between them, in terms of their effectiveness, is their penetration.

UV has limited penetration in air so sterilisation only occurs in a fairly small area around
the lamp. However, it is relatively safe and is quite useful for sterilising small areas, like
laminar flow hoods.

X-rays and gamma rays are far more penetrating, which makes them more dangerous
but very effective for large scale cold sterilization of plastic items (e.g. syringes) during
manufacturing.

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Lab 3

Components of plant culture medium

Medium Preparation

Introduction :

Whole Plants are the only organisms who can provide it's needs internally through the
process of photosynthesis, where it can get the carbon dioxide from the air and water
from the soil (the water contain ) metal elements . using light energy the plant converts
the metal elements to chemical energy used during a series of chemical reactions to be
basic materials(carbohydrates - proteins - to pesticides) and also be hormones,
vitamins, nucleic acids and enzymes, also result in metabolic processes within the
group of plant very important secondary metabolites. And the same is what is going in
for plants or plant parts cultivated inside glass containers lab.

Medium : -

Simply it is the nutrient media that is used in tissue culture where we can develop the
different parts of plant . These plants are cultivated for specific reasons . The target
can be getting the callus ,or continue to unfold and divide until we get the vegetative
growths , or radical growth , or continue until you get the whole plant .

Functions of medium:

• Provide water

• Provide mineral nutritional needs

• Provide vitamins

• Provide growth regulators

• Access to atmosphere for gas exchange

• Removal of plant metabolite waste

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One of the most important factors governing the growth and morphogenesis of plant
tissues in culture is the composition of the culture medium. The basic nutrient
requirements of cultured plant cells are very similar to those of whole plants.

Plant media are generally made up of some or all of the following components:

1) macronutrients, 2) micronutrients, 3) vitamins, 4) amino acids or other nitrogen


supplements, 5) sugar(Carbohydrates), other undefined organic supplements,6)
solidifying agents or support systems, 7) and growth regulators(Plant hormones ).

It is known that the used medium has different forms depending on the aim of use,
there are Solid medium or semi-solid medium which contain Agar . However there is
also the liquid medium which has two kind of medium : 1) Stationary liquid medium 2)
Agitated liquid medium which uses electric vibrator device that remain working through
the whole experiment period .

Each type has its advantages and disadvantages, but if you use liquid medium it is
necessary to grow the plant on a particular bridge or holder of filter paper and
submerged it in the medium then place vegetated part on it .

The Medium Components :

1) Macronutrients :
The macronutrients are the components which the plants need in major or high
quantities , provide the six major elements-nitrogen (N), phosphorus (P), potassium (K),
calcium (Ca), magnesium (Mg), and sulfur (S)-required for plant cell or tissue growth.
The optimum concentration of each nutrient for achieving maximum growth rates varies
considerably among species.

2)Micronutrients
Elements required by the plant but in a very small quantities, which usually does not
surpass a few milligrams .The essential micronutrients for plant cell and tissue growth
include iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum
(Mo).

According to the recommendations of the International Association for Plant physiologist


the elements needed by plants in quantities greater than 0.5 mg / l is classified as
Macronutrients but less than that will be considered as Micronutrients.

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3) Vitamins
Vitamins works as an Assistant in enzymatic systems. They are required in very small
amounts, thiamine (B1), is more commonly used in plant tissue cultures and other
vitamins such as nicotinic acid, pyridoxine (B6) etc.

4) Amino Acids or Other Nitrogen Supplements


Although cultured cells are normally capable of synthesizing all of the required amino
acids, the addition of certain amino acids or amino àacid mixtures may be used to
further stimulate cell growth. The use of amino acids is particularly important for
establishing cell cultures and protoplast cultures. Amino acids provide plant cells with an
immediately available source of nitrogen, which generally can be taken up by the cells
more rapidly than inorganic nitrogen.

5) Carbon and Energy Source


Every living organism needs to have a source of energy in order to complete all the vital
processes within the organism, and therefore each medium needs sugars as a source
of carbon and energy .The preferred carbohydrate in plant cell culture media is sucrose.

6) Solidifying Agents or Support Systems

Agar is the most commonly used gelling agent for preparing semisolid and solid plant
tissue culture media. Agar has several advantages over other gelling agents. First,
when agar is mixed with water, it forms a gel that melts at approximately 60°-100° C
and solidifies at approximately 45°C; thus, agar gels are stable at all feasible incubation
temperatures. Additionally, agar gels do not react with media constituents and are not
digested by plant enzymes.

7) Growth Regulators

Plant growth is the result of the growth of all cells and tissues and organs and thus we
find that there is organic material inside the plant produces inside the plant to regulate
growth and development called natural hormones. These organic materials made in
very small quantities in certain places of the plant and then move to other places to play
it important physiological job. Plant hormones are divided into three groups : Growth
Position, Growth Inhibitors, Growth Activated

The Murashige & Skoog Media is considered to be one of the most famous media used
for propagation of many plant species, which can be prepared in the laboratory or
purchased .

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MS stock solutions

Murashige and Skoog stock solutions

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Lab 4

Micro propagation

Have you ever wonder you how can you take your favorite plant and turned it into many
plants ?! This is the concept behind plant tissue culture

-The term ( plant tissue culture return to the in-vetro cultivation of all plant under aseptic
conditions ..

- an important contribution due this technique is the unique capacity of a single plant cell
to give rise to hole plant this is called Totipotency ..

-using invaluable technique of micropropagation the plant can be then farther expanded
to a infinite number of plants HOW ?

Focusing in plant tissue culture new technique include

1)initiation a culture from a primary Explant

2)shoot initiation and micropropagation

3)rooting

Initiation of culture from a Explant using bean

Explant can be cultured as a mass of poorly differentiated cells called callus

These cells can be manipulated in order to make an entirely new plant by hormones
and media

With the use of any tissue from the outside world the first and most important step in
plant tissue culture is the sterilization of primary tissue.

This means these non-sterilized tissue are surface sterilized and then place in an
appropriate media to stimulate in-vetro growth

Procedure:

- Began with sterilize your work area with 70% Ethanol all surface should be wiped
down

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The supplies you will need in the hood to perform this lab are : green beans seed,
20% bleach, 70% ethanol, sterile beaker, sterile water, sterile jar with a cap, waste
container, non-sterile beaker, sterile petri-dishes, been callus initiation medium
plates, Para film, aluminum foil, Forceps and benzene burner ..

- Poor about 30ml of ethanol in non-sterile beaker containing the beans seeds

- Allow about 60 to 90 Sec exposer to the ethanol (( any longer can kill the seeds ))

Safety warning : when working with the flame and the ethanol together always keep
them a good distance away from each other as ethanol is Extremely flammable

- Dump the ethanol and add about 30ml of sterile water to the beaker .. Dump the
water after shaking

- Using the forceps transfer the seeds to a sterile beaker

- Add about 30ml of bleach (15 min)

- Dump the bleach and wash 3 times with distilled water

- Transfer the seeds to a sterilized jar and add sterile water

Cap loosely and allow the seeds to imbibe all the night ..

Next day

- Wash the seed three times with sterilized water

- Poor the seeds into one of the empty sterilized Petri dishes

use the( frame sterilized cooled ) forceps to remove the seeds coat

- Cut the seed into two parts in a long access ( you should know be able to see the
embryo

- Aseptically transfer the embryo to a callus initiation medium

For this experiment b5 Medium is chosen; contacting nutrient salt vitamins, 30%
sucrose, kynatine(plant hormone) and a gelling agent ( Gelrate )

the PH of the medium 5.8

rap the Petri plate in Para film and then a layer of aluminum foil

incubate the plate in 25 to 27 degrees in darkness for 2 or 3 days

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micropropagation ……

Micro propagation (tissue culture or in-vitro culture) refers to the multiplication of


plants, in aseptic condition and in artificial growth medium. From very small plant
parts like Meristem tip, callus embryos, anther etc.

This tissue culture technique can be used to produce pathogen free plant plant
without going to this low conventional method of breeding

the initiation of shoot from callus is the first step of propagation


shoot initiation from tobacco callus would be used here as an example of how
micro propagation can enable more industrial use of plant tissue culture

in fact in the united state alone more than 120 million plants are produced by
commercial micro propagation each year
the procedure :
- To begin this procedure you should have : sterile water, medium, 95% ethanol,
forceps, benzene burner, sterile Petri dishes, tobacco callus and shoot
development medium
-begin with wiped all surfaces with ethanol
- remove the forceps from ethanol flame and cool the in sterile water ..

- use the forceps to remove a section of the tobacco callus and immediately
place it in sterilized Petri dish and close it

cut the callus into smaller section each about the size of a pencil eraser-

- use the forceps to replace the callus into a dish containing shoot development
medium

- Replace the forceps to the ethanol

in this experiment the medium that have been used is MS media with 3%
sucrose and cytokines ( plant hormone)

- Incubate the culture in 25c in a light to stimulate the shoot formation ( shoots
should be formed whiten two weeks ) the shoot have been initiated and grown
and they can be cell cultured into a new medium to allow more shoot growth

multiplication step make a micro propagation of plant such a powerful tool

to begin prepare your sterile working area


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in this part of the experiment you will need the culture dish with shooting tobacco
callus

dishes of prepared hormone medium of sterile water, 70% ethanol, forceps,


benzene burner.

- Place a sterile petri dishes in front of you

- You use this to cut and transfer tobacco shoot

- Transfer the clump of shoot to the open sterile petri dish

- Cut the mass in half

- Transfer the shoot into fresh medium contain hormones

- Incubate the culture as before under controlled light and temperature condition

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The Difference of the Genomic DNA Extraction Between Animal & Plant

Lab 7

The structure of double-stranded DNA is universal in all living cells, but differences occur in the
methods for extracting genomic DNA from animal and plant cells. Genomic DNA is found in the
nucleus of cells. The amount and purity of extracted DNA depends on the type and size of the
cell, as certain cells contain more DNA and impurities than others.

DNA extraction is one of the most modern of the biological sciences. It is used to diagnose
many medical conditions and can also be used for genetic engineering of both plants and
animals. DNA extraction can also be used to gather evidence in a crime investigation.

Plant Animals
DNA extraction is used in the genetic DNA extraction is also the first step in genetic
modification of plants. Many engineering of animals. Genetic engineering of
agricultural companies use genetic extraction animals is a very broad topic that ranges from
to create DNA that they then modify to make a changing a single gene to make, in an
particular genetic strain of a crop that is example from a Taiwanese research lab, a pig
resistant to various chemicals or pests. An that glows in the dark. On the most complex
example is number of lines of seeds end of the spectrum of animal genetic
manufactured by the Monsanto Corporation engineering is cloning, a process from which
that are immune to the herbicide Roundup. By genetic identical animals can be mad.
making the crops (beets, for example)
resistant to Roundup, that particular herbicide
can be sprayed on fields to kill weeds, but not
affect the beet crop.

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Plant DNA extraction Animal DNA Extraction

Plant genomic DNA is more difficult to extract Peripheral blood leukocytes are a main source
because of the plant's cell wall, which is of animal genomic DNA, but sample collection
removed by homogenization, or by adding is difficult as blood must be withdrawn from the
cellulase to degrade the cellulose that makes animal. Blood contains a range of compounds
up the cell wall. Also, the metabolites present like proteins, lipids, white blood cells, red
in the plant cell may interfere with genomic blood cells, platelets, and plasma, which can
DNA extraction by contaminating the DNA contaminate the DNA sample. The primary
sample during the precipitation process. contaminant of animal DNA extracted from
blood samples is heme, the non-protein
component of hemoglobin.

Differences :

The differences between plant and animal DNA lie in the sequence of bases in the helix.
Compounds found in plant cells are absent in animal cells, and DNA base sequences reflect
this, as the genomic plant DNA is often larger than animal DNA. These differences affect
extraction methods, as it impacts on yield and purity of DNA.

Plant and Animal Cells

Plant cells are distinguishable from animal cells by their rigid cell wall and organelles like the
chloroplast. They also contain proteins and enzymes that play a role in photosynthesis. Some
plant cells are polyploidy, meaning they have more than one copy of each chromosome per cell.
Cellular processes occurring in plants such as photosynthesis produce a range of secondary
metabolites. Animal cells do not have a cell wall, but still need to be treated with chemicals like
sodium dodecyl sulphate (SDS) to disrupt the cell membrane to release genomic DNA.

General DNA Extraction

Plant and animal cells treated with a soapy substance will degrade the lipids in the cell and
nuclear membranes. The DNA mixture will then separate from the cell membranes and proteins.
The DNA in solution can be precipitated using alcohol. Depending on the amount in the sample,
DNA may be visible by the naked eye. Such a simple procedure does not necessarily produce
DNA of high purity.

DNA extraction from a plant

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General way (detergent)
Lab 8

In the next two labs we will extract plant DNA by the general way with soup and salt and with
chemical scientific way with cretin weights and concentrations of used materials .

Extracting DNA from plants with a detergent ( soup ) and salt :

W HY DID I ADD DETERGENT TO MY strawberry?


First blending separated the strawberry cells.

But each cell is surrounded by a the cell membrane DNA is found inside the nucleus within each
cell 0

Why detergent? How does detergent work?

Think about why you use soap to wash dishes or your hands. To remove grease and dirt, right?

Soap molecules and grease molecules are made of two parts:

20
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22
Strawberry DNA Extraction Lesson Plan
This lesson plan is for the extraction of DNA from strawberries. Strawberries are an exceptional
fruit to use for this lesson because each individual student is able to complete the process by
themselves and strawberries yield more DNA than any other fruit (i.e. banana, kiwi, etc.).
Strawberries are octoploid, meaning that they have eight copies of each type of chromosome.
Primary Learning Outcomes
Students will observe first hand that DNA is in the food that they eat. Students will learn the
simple method to extract DNA and why each step is necessary due to the complex organization
of DNA in cells. Students will learn why it is important for scientist to extract DNA from
organisms.

Background:
Strawberries are soft and easy to pulverize. Strawberries have large genomes; they are
octoploid,
which means they have eight of each type of chromosome in each cell. Thus, strawberries are
anexceptional fruit to use in DNA extraction labs.
The soap helps to dissolve the phospholipid bilayers of the cell membrane and organelles. The
salt is used to break up protein chains that bind around the nucleic acids. DNA is not soluble in
ethanol. The colder the ethanol, the less soluble the DNA will be in it. Thus make sure to keep
the ethanol in the freezer or on ice.

Materials and Equipment


For each student: heavy duty ziploc bag (freezer or storage bag); 1 strawberry; DNA extraction
buffer (900mL water, 50mL dishwashing detergent, 2 teaspoons salt); small plastic cup to hold
extraction buffer; cheesecloth to fit in small funnel (4” X 4” should be appropriate); small
funnel; 50mL vial / test tube; glass rod, inoculating loop, or popsicle stick; cold ethanol,
ice.

Procedures/Activities
Step: 1 Duration: 10 minutes
Teacher may choose prior to class to prepare the DNA extraction buffer. In a container add
900mL water, then 50mL dishwashing detergent (or 100mL shampoo), and finally 2 teaspoons
salt. Slowly invert the bottle to mix the extraction buffer.

Step: 2 Duration: 40 minutes to 60 minutes depending on class cooperation


Lab procedures should be conducted as stated in the DNA Extraction: Strawberry lab at the end
of this document. Modifications can be made based on the needs of the students. Some classes
may decide for each student to add individual components of the extraction buffer to the Ziploc
bag (roughly 2 tsp water, 1 tsp soap, 1 pinch salt), while other classes may choose to use the
teacher prepared extraction buffer (from Step 1).
When the students add ethanol to their strawberry extract, they will see the fine white strands of
DNA precipitate. The DNA will form cotton like fibers that will spool onto the stirring
rod/inoculating loop/popsicle stick. then we will store the D A in distil water in 4 C .

Extension
The yield of DNA in this lab may be compared to that of the DNA Banana Extraction lab.
Compare ploidy levels and how it may relate to the amount of DNA recovered. Use varying
concentrations of ethanol (70-100%) to determine how ethanol concentration qualitatively
affects the yield of DNA.

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DNA extraction from a leaf with chemicals
Lab 9

Isolation of total genomic DNA for analysis was carried out avoiding the use of toxic
compound such as phenol or chlorophorm.

Procedure:
1) collect 0.2 gm of banana shoots .
2) ground in 600µl extraction buffer .
3) transfer to new eppendorf tubes.
4) Incubate in water bath at C for 45 min.
5) Centrifuge at 12000 rpm for 10 min.
6) Remove the supernatant and transfer to new eppendorf tubes.
7) Add 150µl ammonium acetate .
8) Incubate at - C for 30 min .
9) centrifuge at 10 000 rpm for 5 min
10) Transfer the supernatant to a new eppendorf tube.
11) Add 1ml of ice cold ethanol .
12) Centrifuge at 12 000 rpm for 5 min .
13) Discard the supernatant .
14) Wash the DNA pellet three times with ice cold Ethanol (70%)
15) Leave the pellets to air dry for a minute.
16) Resuspend the pellets in l steril water and store it at C.

DNA extraction buffer :


100 mM Tris base (pH : 8 ) 12.11 gm/L
50 mM EDTA (pH : 8 ) 18.612 g/L
500 mM NaCl 29.22 g/l
10 mM Mercaptothanol 8.33 ml/l

Ammonium Acetate :
5M Ammonium acetate 38.45gm/100 dist water.

TE buffer:
Tris - base (pH:8) 100mM
EDTA (pH:8) 1mM

Gel loading buffer :


1 ml glecerol + 1 ml dis water + 2% (w/v) Bromophenol blu .

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Running plant DNA extracted by different methods

in Agarose gel .

Lab 10

In this lab we will run the two DNA samples we got in the previous two labs . Recording the
results we will get. Students should learn in this lab how to prepare the Agarose gel, deal with
buffers , loading DNA samples, taking results , writing a scientific lab report.

Agarose gel electrophoresis :

Is a technique that is used to separate charge molecules especially proteins and nucleic
acids as DNA, RNA that differ in size, charge or conformation. It is one of the most widely-used
techniques in biochemistry and molecular biology.

Principle:

Under the influence of electrical field, charged molecules will migrate toward the electrode
that carry an opposite charge. A mixture of different charges molecules will migrate according to
their charges, similar charge compounds will migrate according to their size. Larger molecules
migrate more slowly than smaller ones, and so the distance of migration within a gel can be
used to determine a molecule's size.

Factors that effect on the sample migration:

1. Charge, size, and shape of sample.


2. Gel concentration "pore size".
3. Strength of electrical field.
4. Buffer pH: effect on electrical conductivity.

(-) (-)
- -
- -
-
Power supply Power supply
- -
- -
-
(+) (+)
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Figure 1: Sample is separated according to their charge. Figure 2: Similar charge mixture is separated according to their size.
Requirement of gel electrophoresis:

1- Gel "supporting media".


2- Buffer.
3- Fluorescent dye.
4- Samples
5- DNA Marker.
6- Electrophoresis apparatus: Tank, plate, electrodes, power supply, and combs.
7- Detection system.

1. Gel:
 Agarose:
It is a polysaccharide extracted from seaweed and is typically used at concentrations of 0.5%
to 3%. The higher concentration of agarose is the "stiffer" the gel. Polymerized agarose is
porous, allowing the movement of DNA through it. Agarose gels have a large range of
separation, but relatively low resolving power. DNA fragments from about 0.2 kbp to 50 kbp
can be separated in agarose. Purified agarose is in powdered form, and insoluble in water or
buffer at room temperature but it dissolve in boling water. Different purities of agarose are
commercially available as are agaroses with different melting properties.

2. Buffers:
Two buffers are used together:

Electrophoresis buffer:

Provide ions to conduct the electricity and to maintain the pH at constant value. TBE buffer
(Tris/Borate/Na2EDTA) is usually used.

Loading buffer:

There are many names: Tracking buffer (Tracking dye) and blue juice.

It is used a color marker and density to the sample when load into wells. It is carries slight
negative charges in neutral buffers and thus migrate in the same direction as the DNA during
electrophoresis. For example, bromophenol blue, Xylene Glycerol, or Orange G.

26
3. Fluorescent dye:
It is important to visualize the separated DNA bands, usually ethidium bromide (EtBr) is used.
EtBr is a fluorescent that intercalating between the bases of DNA and glows pink when excited
by UV dye. It is previously mixed with the gel or in tank containing buffer, or after
electrophoresis the gel is soaked in a solution contains EtBr.

4. Samples:
It can be DNA, RNA and Protein.

5. DNA Molecular weight Marker:


There are many names: DNA Molecular weight Marker, DNA ladder, or DNA
standard. It is a mixture of DNA fragments of known sizes. The size of a fragment
is measured by base pairs (bp). Two common ladders are used the 100bp and the
1000bp (1kbp) ladders. Ladders are commercially available by different companies.
The 100 bp ladder is composed of a mixture of small fragments (100 to 2000 bp).
The 1000 bp ladder is composed of a mixture of larger fragments (500 to 12000
bp).

When ladder run in a gel electrophoresis, the fragments will be separated into
distinct bands that can be used as size references.

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6. Electrophoresis apparatus:
 Tank:
It is the container which contain the buffer. It always has a cover to prevent the evaporation
of buffer and for safety. There are two types of tanks: one is called the horizontal while the
other is the vertical. In the horizontal tank we are using agarose or acrylamide gel as a
support media and it is used to identify DNA and RNA. On the other hand, vertical tank only
polyacrylamide gel is used. Vertical is used mainly in identifying protein and in DNA
sequencing.

 Tray: Is the actual mold which provides a shape for the gel as it polymerizes (or solidify).
After polymerization, the gel will move out of the mold and submerse it in a tank of buffer to
run.

 Support: This is just a small piece of glass or plastic that rests snugly in the bottom of the
tray. When the gel is finished polymerizing, the support is gently pushed upwards out of the
tray.

 Power supply: It can be monitored and operated in current (amps), voltage (volts) or power
(watts) mode. The black and red cords leading from the power supply are then attached to
the tray in which the gel is run.

 Combs: It used to make wells on the gel to load different samples.

7. Detection system:
Transilluminator (Ultraviolet light box) and camera: to visualize the bands. After running the
electrophoresis, the gel is placed on a UV light box. It's picture is either acquired by camera so
fragments of sample can be detected.

Which DNA can be used for electrophoresis?

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1. PCR product
2. DNA that cut with restriction enzyme. Then these fragments are separated according to their
sizes.

Result analysis:

A. Band location: It is interested band that indicates the size of DNA fragment when it
compare with the ladder or marker.
B. Intensity of the band: Depends on the DNA concentration.
C. How many fragments: According to the DNA sample and how is it treated (which restriction
enzyme).
D. Other bands: It will be appeared other than interested band such as: primer dimer, non
specific bands, residue in well, salt PCR buffer, protein, or RNA contamination, and smear.

(A) (B) (C)

Band location as PCR product Intensity of the band Fragments cut with different restriction
enzymes separated according to their size.

(D)

29
Detection and analysis of the reaction product

Aim:

We will use agarose gel electrophoresis to show DNA sample and determine if the way of
extraction affects the run of the gel or not.

Instruments & Equipments:

 Micropipetteor rang 0.5-20 l and Yellow tips.


 Electrophoresis apparatus
 Transilluminator
 Digital camera
 Conical flask
 Boling water bath or Microwave oven
 Microcentrifuge tube (Eppendorf tube)
 Parafilm
 Aluminum foil

Materials:

 Agarose powder.
 TBE (Tris/Borate/Na2EDTA) buffer
 Ethidium bromide (10mg/ml)
 6X Loading buffer (bromophenol blue 0.25% (w/v) and sucrose 40% (w/v)).
 Samples (DNA sample, PCR products, PCR products with cut it)

30
Procedure:

Buffer preparation:

10X TBE buffer

Make 0.89 M Tris-Base (108 g/L), 0.89 M Boric acid (55.0 g/L), and 0.02 M (7.44 g/L) of EDTA-
Na2-salt, complete to 1000 ml with distilled water, and then adjust pH to 8.3 (with NaOH).

1X TBE buffer

Take 100 ml form 10X stock solution and complete to 1000 ml with distilled water. Then fill the
tank of electrophoresis and dissolve agarose powder with this buffer.

we used the formula V1 C1 = V2 C2

Gel preparation:

A. Dissolve agarose powder (2%), i.e. 0.7 gm in 300 ml 1X TBE buffer.


B. Melt the gel in a microwave oven or in boiling water bath until completely melted.
C. Cool the solution to about 60°C.
D. Add 1 µl ethidium bromide stock per 10 ml gel solution and mix gently.
to calculate the percent we divide the percentage by 100 then multiply with amount of
volume ex : 2/100 x 300

Pouring the gel:

A. Before pouring, insert the comb on the tray and pour the gel slowly without bubbles and
allow the gel to solidify at room temperature.
B. After the gel solidify, remove the comb to make a wells on the gel.
C. Insert the tray on electrophoresis chamber and cover the gel with 250 ml 1X TBE buffer
(The samples must be in a direction of the (–ve) electrode usually the black one).

Preparing the sample:

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A. Mix 2- h 2-
loading buffer in eppendorf tube or mix on parafilm.
B.
some markers are already become mixed.

Loading the samples:

A. Write the sample and well numbers on you lab report.


B. First load the DNA marker in the first and last well and then load the other samples between
them.
C. Turn on the power supply.
D. After finishing the electrophoresis, transfer the gel to transilluminator, visualize the bands
and then take a photo with camera.

NOTE:

 Always wear protective gloves and eyewear when handle with preparation of gel and
observing DNA on a transilluminator to prevent damage to the eyes from UV light.
 EtBr is carcinogenic (Take precautions)

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how to write a lab report :

Title

When writing the title, the following should be included:

1. The title of the experiment.


2. The students name in full.
3. The instructor or person for whom the lab report is being compiled.
4. The date on which the experiment was performed or the date the lab report was written.

Introduction :

Under this heading should be an overview of what the experiment was about. A sound
definition of what was learned about the process being carried out during the experiment should
be included.

Materials and Methods

This section should contain a description, in the students own words, of the experimental
procedure that was followed in the performance of the experiment. The materials and methods
section should be complete enough so that another student with the same background, but
unfamiliar with the experiment, could perform the same experiment without additional
instructions. Procedures and equipment used should be written in a sentence form. Do not list!

Results

The result section should contain raw data. Raw data consist of actual measured values
recorded during the experiment. Use tables to present this information. All tables should have
descriptive titles, and they should show the units of data entries clearly. The data section
should also contain any graphs that are required. This is an effective method for
communicating experimental results. The following steps should be taken into consideration
while plotting a graph:

1. Do not use tiny dots, use symbols like X or O.


2. Do not draw a series of straight line segments between experimental data points plotted on
a graph. The purpose of many of the experiments is to verify theoretical relationships
between variables.
3. All graphs should have descriptive titles. These titles should tell what the graph is intended
to show. Each axis of a graph should be labeled with the variable and unit it represents.
Always use graph paper and always label graph coordinate lines so that it is easy to see
how
many units each division represents.

33
Discussions and Conclusions

This is the interpretation-and-conclusion of your report. This section should include the
following:

1. How the conduct of the experiment met the objectives.


2. What took place during the process.
3. All questions should be answered within this section in a very logical and clear manner.
The questions should be put into statement form.
4. The conclusions should be relevant to the experiment that was performed and should be
based on facts learned as a result of the experiment.
5. You should also include any recommendations that you feel would improve the experimental
procedure. If you have any further investigations that might be suggested by the data, you
should also include them here.

34
Detection of Genetically Modified Foods by PCR

Lab 11

Learning objectives….

 Upon completion of this lab students should understand and be able to….

◦ The relationship between genotype and phenotype.

◦ PCR based detection of GMO modified foods.

◦ Methods for producing transgenic crops.

Laboratory Objectives…..

◦ Carry out laboratory methods for extracting and purifying DNA from plant and
foodstuffs.

◦ Successfully set up and carryout a PCR based detection for the presence of a
GMO transgene.

◦ Understand the purpose of negative and positive control reactions.

◦ Analyze and interpret data obtained from gel electrophoresis of PCR products
from GMO PCR detection reactions.

Introduction to GMO Foods…

 First “green revolution”

◦ Took place 1950s-1970s

◦ High yield strains of crops

◦ Extensive high tech agriculture methods

◦ Greatly increased world food supply

◦ Most dramatic in developing nations

◦ Limitations of this revolution

 Toxicity

 No venue for alteration of nutritional content

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Second “green revolution”

 Fueled by genetic engineering

 Genes encode traits such as: herbicide resistance, insect resistance, drought
resistance, frost resistance, nutritional factors, and others.

 Examples include, golden rice and roundup ready crops.

 Public concerns about labeling and safety.

Overview of Transgenic Process…

36
Molecular Transgenic Process…

Common Transgenes….

 Transgenes defined as the “transferred gene”

 Bt gene from Bacillus thuringiensis

◦ Produces a toxin against caterpillars

 Glyphosate resistance gene

◦ Confers resistance against Roundup herbicide

Prevalence of GMO foods in the US

 Greater that 60% of fresh vegetables and processed foods sold in supermarkets today
are genetically modified.

 In 2004 ~85% of soy and 45% of corn were grown form RR seed.

 Currently no US federal regulations require declaration of genetically modified foods.

 Situation is different in EU.

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Detection of GMO Foods

 PCR based detection provides rapid diagnostic.

 Primers specific to promoter driving the transgene.

 35 S promoter present in many transgenic plants.

Overview of Laboratory Exercise

 Prepare DNA from foodstuffs

 Set up PCR reaction

 Run PCR products on 2% gel

 Analyze PCR products and assess results

38
February 24, 2009

MDT_Report #2_Lab 5_GMO – DNA detection by PCR.

Tai Hoang – BT.410

INTRODUCTION

Transgenes that expressed in known and unknown food samples can be readily detected by using PCR –
reactions and appropriate primers/promoters.

During this lab session, we will attempt to identify transgenes expressed in GMO food products using
DNeasy Kit for DNA purification and Agarose Gel Electrophoresis to detect genes of interest in Roundup-
Ready leaf (+ control), wild-type seeds (- control), and food product (Kix cheerios).

MATERIALS

Materials Purpose Quantity


DNeasy Blood & Tissue Kit Isolation of DNA of interest 1
Ready-To-Go™ PCR Analysis Beads (consisted of PCR reaction-ready beads 6 micro-
thermostable Taq®, dNTPs, BSA, MgCl2, and pH8.3 tubes
buffer containing Tris and salts.)
35S primer Common primer/promoter found in wide ~ 70uL
range of GMO products.
Tubulin Primer House-keeping genes ~ 70uL
Samples (R.R. leaf, WT seeds, food) Minus amounts used as +control, -control,
and unknown sample, respectively.
Buffer ATL Lysis buffer 180uL
Proteinase K 20uL
Buffer AL Denaturing buffer 200uL
Ethanol (96 – 100%) Precipitation of DNA 200uL
Buffer AW1 Washing 500uL
Buffer AW2 Washing 500uL
Buffer AE Elution buffer 100uL
Thermocycler PCR reaction
2% Agarose Gel set-up at 50mL volume in 1X TBE Gel Electrophoresis
buffer
pBR322/BstN1 color marker Ladder 12uL
Standard Lab reagents
Standard Lab Equipments

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METHODOLOGY

Isolation of DNA from food products: (using QIAGEN DNeasy® Blood & Tissue protocols as reference)

1. Obtain (6) 1.5mL micro-tubes and label accordingly:


a. Tube #1: R.R/leaf/35S/date/name; (R.R = Roundup-Ready, leaf = DNA used as +control,
35S = promoter for the expression of transgenes).
b. Tube #2: WT/seed/35S/date/name; (WT = Wild-type, seed = DNA used as –control).
c. Tube #3: Food/35S/date/name; (Food = unknown sample to be analyzed for transgene).
d. Tube #4: R.R/leaf/T/date/name; (R.R = Roundup-Ready, leaf = DNA used as +control, T =
promoter for the expression of house-keeping genes).
e. Tube #5: WT/seed/T/date/name; (WT = Wild-type, seed = DNA used as –control).
f. Tube #6: Food/T/date/name; (Food = unknown sample to be analyzed for transgene).
2. Obtain (6) samples in small quantities for (6) tubes and place in corresponding tubes. Grind each
sample thoroughly into tiny particles.
3. To each tube, add 180uL Buffer ATL and 20uL proteinase K. Vortex briefly to mix.
4. Incubate at in 56 degree C water-bath for 1hr. Vortex occasionally to disperse samples during
incubation.
5. Prepare an Agarose gel set-up in meantime: (2% Agarose, 50mL Gel, 1X PBS, and 2.5uL EtBr).
6. After incubation, vortex again for 15 seconds. (Refer to DNeasy Kit protocol for remaining steps
until running PCR reactions).
7. To each tube, add 200uL Buffer AL. Vortex to mix.
8. Add 200uL ethanol (96 – 100%). Vortex to mix.
9. Spin for 1 min. at 8,000 rpm.
10. Transfer supernants only to set of (6) new micro-tubes. Label accordingly.
11. Wash with 500uL Buffer AW1. Spin for 1 min at 8,000 rpm.
12. Wash with 500uL Buffer AW2. Spin for 3 min at 14,000 rpm.
13. Transfer spin columns to new set of collection tubes (6 new micro-tubes)
14. Add 100uL Buffer AE (Elution Buffer) directly onto DNeasy membrane at bottom of spin column.
15. Allow to stand at RT for 1 min to incubate.
16. Spin for 1 min at 8,000 rpm. Collect and retain the supernant flow-through in the 6 micro-tubes
labeled as previous.
17. Place on ice until PCR set-up is ready.

40
PCR reactions:

1. Use table below to prepare samples for PCR reactions: Use (6) micro-tubes containing Ready-to-
go Bead to prepare the samples.

Tube Label 35S primer DNA Tubulin Total Vol.


1 R.R/leaf/35S 22.5uL 2.5uL - 25uL
2 WT/seed/35S 22.5uL 2.5uL - 25uL
3 Food/35S 22.5uL 2.5uL - 25uL
4 RR/leaf/Tubulin - 2.5uL 22.5uL 25uL
5 WT/seed/Tubulin - 2.5uL 22.5uL 25uL
6 Food/Tubulin - 2.5uL 22.5uL 25uL

2. Place all tubes in Thermocycler at settings appropriate for PCR reactions.


3. Running the PCR reactions for ~ 2hrs.

Gel Electrophoresis:

1. Load 12uL of pBR322/BstN1 to well #7 from the left of desirable gel.


2. Load 12uL of each sample (1 thru 6) to corresponding wells in a subsequence order, i.e. from
well #1 thru well #6. (NOTE: samples contain primers mixed with dye, additional dye is not
necessary).
3. Make note/table for location of each sample and gel number.
4. Run gel for 30 mins at 120 Volts.
5. Analyze band patterns after electrophoresis and record all results in lab notebook.

41
RESULTS and DISCUSSIONS

Gel Photo: (Gel #5)

1. According to the gel loading order, results for lane #1 thru lane #6 are as followed:
a. Lane #1: Single light band with a touch of smear – Transgene present.  +control
sample used.
b. Lane #2: No visible distinct band, only a smearing streak – No transgene.  Negative
control sample containing no 35S promoter. Smearing pattern might indicate nucleic
acid contaminants.
c. Lane #3: Single distinctive band visible – Transgene present.  Food product contains
GMO materials.
d. Lane #4: No visible band – Indication of error.  All samples containing Tubulin house-
keeping genes should show distinct bands since the genes are being constituently
expressed.
e. Lane #5: Distinct band with smearing – Indicating presence of Tubulin gene and DNA
overload.
f. Lane #6: Single visible band – Presence of Tubulin.  A positive result of GMO detection
in food product.
2. A negative result is observed when either the 35S promoter was not present or bad technique(s)
were employed while performing various procedures.
3. To improve possible results when repeating the experiment, care must be taken when preparing
and loading the samples, as well as when performing DNA isolation steps.

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CONCLUSION

Identification of gene of interest is another application of PCR amplification reaction. By using


commercially available kits and reagents, targeted genes can be easily isolated, purified, and detected,
provided that the experimental protocols must be precisely followed and the basic laboratory
techniques must be mastered.

43