2. Background
Genes to identify: 35S promoter, NOS terminator, and
PSII
Methodology
a) DNA Extraction
b) DNA Amplification
c) Electrophoresis of DNA
Analysis of Results
Conclusion
3. GMO’s are organisms that have been modified through
genetic engineering.
In the United States, under guidelines issued by USDA's
Animal and Plant Health Inspection Service, genetic
engineering is defined as the genetic modification of
organisms by recombinant DNA techniques (USDA, 2007)
According to the USDA, crops have been genetically
modified ever since 1996.
These crops have all kind of modifications, such as:
herbicide tolerance, pesticide tolerance, and bacterial
resistance.
5. Steps for genetic
modification of a
crop:
a) First, find the
ideal protein to
express.
b) Isolation of the
gene of interest.
c) Insertion of the
ideal promoter
and terminator
d) Back cross to
highest-yielding
plants
6. 35S promoter from the cauliflower mosaic
virus (CaMV 35S)
Nopaline synthase (NOS) terminator from
Agrobacterium Tumefaciens
Photosystem II (PSII)
7. Our hypothesis is that the genes 35S and NOS
will appear in the gel. The 35S gene will be
found at 203bp and NOS at 225bp. We
believe the corn will be identified as GMO
while the soymilk won’t. In addition, both the
corn chips and the papaya will be indentified
as GMO.
8.
9.
10. DNA Extraction
a) Weighing and
Grinding of samples
b) Pipette 50µL of Slurry
into tube containing
Instagene matrix.
c) Flick tube and place
in a 95 degrees
celsius water bath for
5 min.
d) Place tubes in
microcentrifuge for 5
min.
Non-GMO Test food Test food
50µL of
sample
slurry
500µL of
Instagene
matrix
50µL of
sample
slurry
500µL of
Instagene
matrix
50µL of
sample
slurry
500µL of
Instagene
matrix
11.
12. PCR
a) 20µL of indicated
master mix is
dispensed to each
of the samples.
b) 20µL of DNA
samples are
dispensed to their
corresponding
tubers.
c) Once the samples
have been
dispensed, the
tubes are put in
the thermal cycler.
Non-GMO PMM
Non-GMO GMM
GMO (+) PMM
GMO (+) GMM
Test PMM
Test GMM
Test PMM
Test GMM
PMM= Plant molecular marker
GMM= Genetic molecular
marker.
13. Electrophoresis
a) The gel used was a 3% Agarose
gel.
b) To prepare for electrophoresis,
10µL of Orange Loading dye was
deposited into each of the
samples.
c) 20µL of Molecular weight
ruler(mwr) was deposited to wells
to wells 1 and 10.
d) 20µL of samples were dispensed
to wells 2-9.
e) Electrophoresis was run for
30min. at 100V.
f) After electrophoresis, the gel was
stained with ethidium bromide
pads for 20min.
1 Molecular weight Ruler
2 Non-GMO PMM
3 Non-GMO GMM
4 GMO (+) PMM
5 GMO (+) GMM
6 Test PMM
7 Test GMM
8 Test PMM
9 Test GMM
10 Molecular Weight Ruler
17. One can conclude that the maize was
genetically modified, while the soymilk,
papaya, and corn chips are inconclusive.
The experiment must be repeated because
the results were affected by human errors,
such as bad pipetting technique.
Notas do Editor
Main Entry: re·com·bi·na·tion
Pronunciation: \ˌrē-ˌkäm-bə-ˈnā-shən\
Function: noun
: the formation by the processes of crossing-over and independent assortment of new combinations of genes in progeny that did not occur in the parents
InstaGene™ matrix: What Function Does It Perform?
InstaGene matrix consists of a suspension of negatively charged, microscopic beads
that bind divalent cations such as magnesium (Mg2+). It is important to remove divalent
cations from the extracted DNA samples because the cations assist enzymes that degrade
the DNA template. When cheek cells are lysed and boiled in the presence of InstaGene
matrix, the divalent cations released from the cells bind to the beads, and the heat inactivates
the DNA-degrading enzymes. The beads are pelleted by centrifugation, and the supernatant,
which contains clean, intact genomic DNA, can be used as template in PCR reactions.
The beads in the InstaGene matrix quickly settle out of the suspension. It is therefore
extremely important that the vial of matrix be thoroughly mixed before pipetting aliquots for
each student workstation, so that the aliquots contain equivalent amounts of beads.
If the DNA samples are not going to be amplified within 24 hours, they can be stored in
the refrigerator in the InstaGene matrix for up to 1 week. For longer storage, place samples
in the freezer to prevent DNA degradation. Before the samples are used in PCR, the beads
should be repelleted by centrifugation just prior to making up the PCR reactions.
Master Mix: What Is It?
The master mix contains a mixture of nucleotides, or dNTPs (dATP, dTTP, dCTP, and
dGTP), buffer, and Taq DNA polymerase. Complete master mix is prepared by adding
primers to the master mix just prior to the laboratory period. When 20 μl of the DNA template
is added to 20 μl of complete master mix, all of the necessary components for a 40 μl PCR
reaction are present.
Note: Once the master mix and primers are mixed, the complete mix should be kept on ice
and used within 30 minutes to 1 hr. These reagents are extremely sensitive.
Why Are the Primers Red and Green?
The primer mixes contain PCR-compatible dyes that allow students to easily visualize
and distinguish the different master mixes. The dyes also migrate in the gel giving a visual
demonstration of electrophoresis.