Documente Academic
Documente Profesional
Documente Cultură
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
2
The Final Exam
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Lab 1
Safety and regulation
What is safety?
Elimination of potential threats to human health
- Hazard means the equipment, chemicals and conditions that have a potential to cause
harm such as chemicals ,electricity, animals and infectious agent .
- 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.
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- Environmental protection
- The use and handling animals
- Regulation of radioisotopes.
2- Institutional Responsibility.
- 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.
• 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.
• Minimize use of sharp objects and be sure that you properly dispose of them.
• Use fume hood for any chemicals or solvent that you can smell.
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Lab 2
Many agents and procedures have been developed to accomplish this end.
A. Generally, decontamination involves physical and/or chemical agents.
Chemical decontamination agents are substances that react with and thus alter some
important molecular component of the cell.
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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.
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Sterilization techniques:
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.
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 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.
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
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
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 vitamins
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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:
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 .
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).
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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.
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
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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 ?
3)rooting
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
Cap loosely and allow the seeds to imbibe all the night ..
Next day
- 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
For this experiment b5 Medium is chosen; contacting nutrient salt vitamins, 30%
sucrose, kynatine(plant hormone) and a gelling agent ( Gelrate )
rap the Petri plate in Para film and then a layer of aluminum foil
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micropropagation ……
This tissue culture technique can be used to produce pathogen free plant plant
without going to this low conventional method of breeding
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
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
- 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 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.
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.
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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 .
But each cell is surrounded by a the cell membrane DNA is found inside the nucleus within each
cell 0
Think about why you use soap to wash dishes or your hands. To remove grease and dirt, right?
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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.
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.
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.
Ammonium Acetate :
5M Ammonium acetate 38.45gm/100 dist water.
TE buffer:
Tris - base (pH:8) 100mM
EDTA (pH:8) 1mM
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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.
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.
(-) (-)
- -
- -
-
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:
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.
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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.
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.
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.
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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.
Band location as PCR product Intensity of the band Fragments cut with different restriction
enzymes separated according to their size.
(D)
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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.
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)
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Procedure:
Buffer preparation:
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.
Gel preparation:
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).
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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.
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
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.
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:
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Discussions and Conclusions
This is the interpretation-and-conclusion of your report. This section should include the
following:
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Detection of Genetically Modified Foods by PCR
Lab 11
Learning objectives….
Upon completion of this lab students should understand and be able to….
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.
◦ Analyze and interpret data obtained from gel electrophoresis of PCR products
from GMO PCR detection reactions.
Toxicity
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Second “green revolution”
Genes encode traits such as: herbicide resistance, insect resistance, drought
resistance, frost resistance, nutritional factors, and others.
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Molecular Transgenic Process…
Common Transgenes….
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.
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Detection of GMO Foods
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February 24, 2009
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
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METHODOLOGY
Isolation of DNA from food products: (using QIAGEN DNeasy® Blood & Tissue protocols as reference)
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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.
Gel Electrophoresis:
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RESULTS and DISCUSSIONS
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
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