TECHNIQUES IN MOLECULAR BIOLOGY – GMO PCR EXPERIMENT
This lab will introduce you to DNA isolation in plant and food product and a straightforward PCR
technique to identify elements of GMO foods.
Background: GMO crops are altered in several ways improve the function of the organism, to
survive in the presence of an herbicide, to reduce pests from destroying the crop and may other
traits. In simple terms a gene or suite of genes are inserted into a plant creating a genetically
modified organism. One of the issues with creating a GMO is ensuring the gene is expressed in the
right tissue of the plant and the control of its expression. Some genes need to be constitutive others
under various plant hormonal control.
One method to ensure proper expression is to clone the gene of interest under the control of an
appropriate promoter and terminator. The most common promoter used in GM crops is the 35S
promoter from the cauliflower mosaic virus (CaMV 35S). This promoter is choses because it has a
native ability to activate transcription in all plant cell types. The most common terminator used in
GM crops is the nopaline synthase (NOS) from Agrobacterium tumefaciens. One or both of these
genetic elements are present in ~85% of all GMO crops currently approved around the world.
You will use PCR to detect the absence or presence of CaMV 35S (203bp) and NOS (225 bp) in two
types of corn chips - unmarked popular brand and a brand identified as organic or GMO free. A
control of plant PSII primers will be used as one of your controls. PSII primers will amplify a region of
455 bp of the photosystem II chloroplast gene.
Laboratory: In your introduction section include a serious introduction to the promoter and
elements that we will identify using PCR. ALSO include a complete protocol for the experiment
(DNA isolation and PCR set up), gel preparation and gel visualization with a small section on the
proposed results and what to expect. For each step you can simply copy the instructions into your
book, but think about the important points as you do so. ALSO include a description of the positive
and negative controls. What are they and why are the necessary?
DNA Extraction. For each sample assemble the extraction as described below. Each group will
extract DNA from TWO samples. Your group will chose from the following: a certified GMO free
sample (instructor provided), a commercially available, student supplied GMO free or organic corn
chip sample or a non-labeled national corn chip brand (instructor supplied).
1. Add 500 µl well suspended InstaGene Matrix to a provided screwcap microfuge vial
(supplied).
• ENSURE the beads are WELL suspended by mixing prior to pipetting.
2. Transfer 40 ml of milliQ water into a clean falcon tube labeled water.
3. Place water tube into 95oC waterbath
4. Mass ~1 g of your "negative" GMO food FIRST (avoid potential contamination with second
GMO containing sample) and place in cleaned mortar. Record the mass.
5. Add 5 ml of warm water per g of sample.
6. Grind with pestle for 2 min until consistent slurry is formed.
7. Add an additional 5 ml of warm water for every gram of food. Continue to mix and grind
until the slurry can be pipetted.
8. Transfer 50 µl of slurry to InstaGene. Mix by vortexing and incubate in 95oC waterbath for 5
min. At the end of the incubation, place mixture on ice while setting up PCR reaction.
9. Wash the morter and pestle with detergent, rinse with water, rinse with 10% bleach and
water to minimize contamination. Always start with the negative control to avoid PCR
detection of positive sample
1
PCR Reaction:
1. Prepare Plant Master Mix (PMM)
• Transfer 64 µl master mix to a tube label PMM add 1.5 µl plant PSII primers, vortex and
briefly centrifuge and store on ice.
2. Prepare GMO Master Mix (GMM)
• Transfer 64 µl master mix to a tube label PMM add 1.5 µl GMO primers (both promoter
and terminator), vortex and briefly centrifuge and store on ICE
3. Number six PCR tubes and include your lab group initials. CHANGE tips each time!\
• Start with Master Mix to all tubes,
• Centrifuge InstaGene beads/chip slurry for 5 min at max rpm. Then add the DNA. DO
NOT TRANSFER any beads to your PCR reaction mixture.
Tube
1
2
3
4
5
6
20 µl DNA
Non-GMO food
Non-GMO food
GMO food
GMO food
GMO pos control DNA
GMO pos control DNA
20 µl Master Mix
Plant master mix (PMM)
GMO Master Mix (GMM)
Plant master mix (PMM)
GMO Master Mix (GMM)
Plant master mix (PMM)
GMO Master Mix (GMM)
4. Subject the mixture after mixing and a brief centrifugation to PCR
Step
Initial Denaturation
PCR amplification
Final Extension
Hold
Function
Denature
Denature
Anneal
Extend
Extend
Temp
94oC
94oC
59oC
72oC
72oC
4 oC
Duration
2 min
1 min
1 min
2 min
10 min
Indefinite
Cycle
1
40
1
1
Gel Electrophoresis:
1. Molecular Weight (ladder) Ruler
• Mix 15 µl BioRad PCR molecular weight ruler with 20 µl of BioRad Orange G loading dye.
Mix and pulse centrifuge.
2. To each PCR reaction, add 10 µl of Orange G loading dye. Mix and pulse centrifuge.
3. Prepare an appropriate agarose gel. Review your notes and handouts for the sizes
predicted for your experiment. Hint, read the intro!
• Don't forget the stain!
4. Load 20 µl of either standard or reaction mixture into each well. DO NOT double mix
standard with PCR reactant.
5. Perform electrophoresis...
Results and Conclusions: In your results/discussion section of the lab book include the following:
• AFTER the experiment is done, insert your figure, determine the size of each band and interpret your results for
each sample.
• A description of each component and what role it plays, why is it important, what does it do?
• Describe with some detail ONE GMO gene (besides bt toxin) and the promoter and terminator used in this
experiment.
• Is your sample GMO free.
• What are the controls used in this experiment and why are they important?
• We are performing duplex PCR. What is duplex PCR?
2