G-Mod Lab Activity
Detecting Genetically Modified
Foods
1. Review the use of micropipettes
2. DNA extraction from food
samples using gene releaser and
temperature cycle
3. Prep PCR components using
extracted DNA from #2
4. Run the PCR program
5. Prepare the samples for
loading
6. Load gels
7. Run gels
8. Analyze gels
Micropipetting
1.
2.
3.
4.
Set micropipette to proper
amount – be sure you are using
the correct volume micropipette
(.5-10uL, 2-20uL, 20-200uL or 40200uL)
Lock the setting once the desired
volume has been located.
Obtain a pipette tip from the tip
box
Depress the push button and stop
at the first stop
Micropipetting - continued
5. Place the tip into the sample to be drawn up
6. Release the push button slowly to draw up
the sample
7. Position the pipette tip in the proper tube
8. Depress the push button all the way to expel
the liquid
9. Remove the micropipette from the tube
10. Release the micropipette push button
* Be sure to change tips between each
solution
Practice Micropipetting
In your group of two, use the two
micropipettes at your station to
measure the following volumes of
solutions. Each person should do each
volume. Be sure to change tips
between solutions
1. 100 uL
2. 4 uL
Protocol for DNA extraction from
sample using GeneReleaser
1. Each group of 4 will select one of the food
sources
2. Proceed to the Student Procedures (p. 27 –
bottom right of page) and begin with #1,
work through number 9 – work through
these steps together
We will all finish together and begin the
temperature cycle to extract DNA – 20
minutes
DNA Extraction using Gene Releaser
• 1. Label the top of the sample tube with your
initials – clear top tubes
• 2. Crumble food sample into powder in sterile lab
tissue paper. Transfer a small amount of food
into sample tube – approximately one pinky
• 3. Add 500 uL of distilled water to the sample and
mix using the plastic disposable pestle
• 4. Centrifuge for 1 minute to obtain a clear
supernatant – Be sure to balance tubes in
microcentrifuge
• 5. If no supernatant is present, add 100
additional uL of distilled water, mix by
inverting the tube and pellet undissolved
materials by centrifugation for 1 minute.
• 6. If supernatant is present, label one of the
small clear 200 uL tubes from the kit with your
initials
• 7. Pipette 50uL of GeneReleaser up and down
within the GeneReleaser tube from the
student kit to mix the contents thoroughly.
Transfer 50uL of GeneReleaser into the newly
labeled clear 200uL tube
• 8. Transfer 10uL of the supernatant from step
5 into the 200uL tube that now contains the
50uL of GeneReleaser. Mix gently with the
micropipettor, being careful to leave all the
liquid in the tube when you remove the
micropipettor
• 9. Place the tube in the MyCube Thermal
Cycler – record your position on the table at
the cycler
GeneReleaser Temperature Cycle
• The GeneReleaser Temperature Cycle will
run for 20 minutes. This is NOT a
polymerase chain reaction process – It is
a program of varying temperatures for
the extraction of DNA
• Once the DNA has been extracted, we
will prepare the sample for PCR
amplification
PCR – Amplification of DNA
• 1. Label the small colored tube found in the
student kit with your initials. The 0.5 mL
screw cap microcentrifuge tube (purple top)
contains the chemicals needed for the PCR
amplification of the plant and GMO vector
DNA. This PCR master mix is in powdered
form and needs to be mixed with water. Add
90uL of distilled water and mix the contents
well by inverting the tube 10 times.
• 2. One partner from each pair should transfer
42uL of the reconstituted PCR mix into their
labeled small colored tubes.
• 3. Obtain the tube containing the GeneReleaser
and extracted DNA. If the GeneReleaser chemical
comes into contact with the PCR master mix, it
will interfere with the amplification process. To
prevent this from occurring, the tube must be
centrifuged to pellet any remaining
GeneReleaser. Place the clear small tubes
containing GeneReleaser and extracted DNA into
the emptied distilled water tube. Make sure
smaller tubes are capped tightly. Run samples for
2 mintues
• 4. Once centrifuged, carefully remove the
smaller tube from the larger one and transfer
8uL of clear supernatant, containing the DNA,
into the colored tube containing the water
and PCR mix. Make sure not to transfer any of
the GeneReleaser (the white material at the
bottom of the tube).
• 5. Place the tubes in the MyCube. Record the
location of the tubes.
• Begin PCR Amplification
What are genetically modified
organisms? (GMOs)
• GMOs are organisms in which the
genetic material (DNA) has been
altered or modified through a
method that does not occur in
nature – this is often done to
introduce a new or altered
characteristic to the modified
organism
Why develop GMOs?
• Modification of agriculturally important crops
For example: Bacillus thuringensis produces a
chemical that is toxic to some crop-damaging
insects, but not to beneficial insects
Roundup Ready crops – Agrobacterium
tumifaciens – produces a protein that
introduces resistance to the killing effect of
Roundup – weeds are still killed and crops are
immune to the effects
How to create a GM plant
What are the potential benefits
of GM products?
• Crops – enhanced taste and quality, reduced
maturation time, increased nutrients, yields and
stress tolerance, improved resistance to disease,
pests and herbicides
• Animals –increased resistance, productivity and
hardiness, better yields of meat, eggs and milk
• Environment – “friendly” bioherbicides and
bioinsecticides, conservation of soil, water and
energy, better natural waste management
Why is there controversy over
GMOs?
• Potential human health impacts – allergens,
unknowns
• Domination of world food production by a few
companies
• Tampering with nature by mixing genes
among species
• Labeling is not mandatory in some countries
(U.S.)
Why test for the presence of
GMOs?
• Labeling standards vary :
U.S. – any product containing less
than 5% GMO may be labeled as
GM free
Europe – will not accept imports
unless they are 0.9% GM or less
What is PCR?
• Molecular copy machine – copies a specific
region of DNA
• 40 cycles:
Temperature is increased to 95 C – DNA strands
separate – denature
Temperature is cooled to between 50-60 C –
annealing – primers bind to DNA
Temperature is increased to 72 C, activating
polymerase, adding nucleotides to the ends of
the primers and extending a new copy of DNA
strand
• Primers used in the PCR
reaction:
• NPTII gene marker – for GMO
detection
• 16S plant ribosomal marker –
control primer
• PCR Virtual lab
What has been amplified and
what are we looking for?
• 1. NPTII – gene that is part of the vector
used to create many genetically modified
organisms – it is 254 base pairs in length
• 2. The second gene is a 16S ribosomal
gene associated with plant chloroplast
DNA – this serves as a control – because
it is present in plants – the amplified
portion of the 16S ribosomal gene is 525
base pairs in length
How does electrophoresis work?
• Gel electrophoresis
Flash Gel Analysis
• 1. Obtain a small tube containing 2uL of
loading dye .
• 2. Transfer 10 uL of the PCR sample (colored
tube) into the tube containing the loading dye.
Mix well by pipetting up and down a few
times in the tube.
• 3. Pipette 10 uL of the PCR/loading dye
combination into one well of the Flash Gel
cassette – record location – see page 29, #3
There are 13 wells in the gel
– Well #1 contains a DNA
ladder, be sure to record
the names of the groups
that have loaded the
remaining wells (p. 31)
Lane 1 = DNA Ladder
Lane 2 = Cheetos
Lane 3 = Cheetos
Lane 4 = Teddy Bear cookies
Lane 5 = Teddy Bear cookies
Lane 6 = Lay’s chips
Lane 7 = Lay’s chips
Lane 8 = Funyuns
Lane 9 = Empty
Lane 10 = Positive
control