Professional Documents
Culture Documents
Laboratory Manual of
Plant Biotechnology
Part 1
Dr. Jehan Salem
I. Bayan Sajer
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Physical and
Chemical sterilization
Components of plant
culture medium
Medium Preparation
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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.
- 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.
Lab 2
Physical and Chemical sterilization
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.
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
dont stand a chance.
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.
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, its 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.
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 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:
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 .
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 acid 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.
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
DNA extraction is used in the genetic
modification of plants. Many
agricultural companies use genetic extraction
to create DNA that they then modify to make a
particular genetic strain of a crop that is
resistant to various chemicals or pests. An
example is number of lines of seeds
manufactured by the Monsanto Corporation
that are immune to the herbicide Roundup. By
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.
Animals
DNA extraction is also the first step in genetic
engineering of animals. Genetic engineering of
animals is a very broad topic that ranges from
changing a single gene to make, in an
example from a Taiwanese research lab, a pig
that glows in the dark. On the most complex
end of the spectrum of animal genetic
engineering is cloning, a process from which
genetic identical animals can be mad.
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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.
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 :
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5M Ammonium acetate
Ammonium Acetate :
38.45gm/100 dist water.
TE buffer:
100mM
1mM
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(-)
-
(-)
-
Power supply
Power supply
-
(+)
Figure 1: Sample is separated according to their charge.
(+)
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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.
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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.
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)
(D)
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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
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)
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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 EDTANa2-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 60C.
D. Add 1 l 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).
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A. Mix 2-
h 2-
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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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.
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.
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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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Genes encode traits such as: herbicide resistance, insect resistance, drought
resistance, frost resistance, nutritional factors, and others.
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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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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 RoundupReady leaf (+ control), wild-type seeds (- control), and food product (Kix cheerios).
MATERIALS
Materials
DNeasy Blood & Tissue Kit
Ready-To-Go PCR Analysis Beads (consisted of
thermostable Taq, dNTPs, BSA, MgCl2, and pH8.3
buffer containing Tris and salts.)
35S primer
Tubulin Primer
Samples (R.R. leaf, WT seeds, food)
Buffer ATL
Proteinase K
Buffer AL
Ethanol (96 100%)
Buffer AW1
Buffer AW2
Buffer AE
Thermocycler
2% Agarose Gel set-up at 50mL volume in 1X TBE
buffer
pBR322/BstN1 color marker
Standard Lab reagents
Standard Lab Equipments
Purpose
Isolation of DNA of interest
PCR reaction-ready beads
Quantity
1
6 microtubes
~ 70uL
~ 70uL
180uL
20uL
200uL
200uL
500uL
500uL
100uL
Denaturing buffer
Precipitation of DNA
Washing
Washing
Elution buffer
PCR reaction
Gel Electrophoresis
Ladder
12uL
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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-togo Bead to prepare the samples.
Tube
1
2
3
4
5
6
Label
R.R/leaf/35S
WT/seed/35S
Food/35S
RR/leaf/Tubulin
WT/seed/Tubulin
Food/Tubulin
35S primer
22.5uL
22.5uL
22.5uL
-
DNA
2.5uL
2.5uL
2.5uL
2.5uL
2.5uL
2.5uL
Tubulin
22.5uL
22.5uL
22.5uL
Total Vol.
25uL
25uL
25uL
25uL
25uL
25uL
Gel Electrophoresis:
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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 housekeeping 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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