Testing For Genetically Modified Foods

1. Extraction of DNA From Food Samples

1. Add 500 µL of purchased PCR mix matrix to each of the 2 screw-cap tubes using a transfer pipet or 200-1,000 µL adjustable-volume micropipette. Pipette up and down with the pipet between each aliquot to evenly mix the PCR matrix.
2. Label one screw-cap tube “non-GMO” and the other “test”.
3. Weigh out 0.5 g of certified non-GMO food and put it into the mortar.
4. Add 2.5 mL of distilled water and grind with pestle for 2 min to form a slurry.
5. Add another 2.5 mL of distilled water and continue grinding with pestle until slurry becomes smooth enough to pipet.
6. Pipet 50 µL of ground slurry to the screw-cap tube containing 500 µL of PCR premix labeled “non-GMO” using the 50-µL mark on a graduated pipet. Recap tube.
7. Repeat steps 1.3–1.6 to prepare the test food sample.
8. Pipet 50 µL of ground test food slurry to the screw-cap tube labeled “test”. Recap tube.
9. Vortex the non-GMO food and test food PCR tubes for 1 min and place tubes in 95 °C water bath for 5 min. If no vortex is available, flick the tube several times to mix before placing in water bath.
10. Place tubes in a centrifuge for 5 min. A solid pellet should form at the bottom of the tube. If pellet is not formed after 5 min, centrifuge again for 2 min intervals until pellet forms.
11. Tubes can be used immediately for PCR or stored in a refrigerator for up to 1 week.

2. Setting Up PCR Reactions

1. Number PCR tubes 1–6 and initial them. The numbers should correspond to the following tube contents listed in Table 1.
2. Place each labeled PCR tube in a microtube holder with caps open.
3. Using a fresh tip for each addition, add 20 µL of the primer indicated on Table 1 to each PCR tube. Cap tubes.
4. Using a fresh tip for each tube, add 20 µL of the DNA sample indicated on Table 1 to each PCR tube, being sure to pipette only from the supernatant and avoiding the solid pellet at the bottom of the tubes.
5. After each DNA sample is pipetted into the corresponding PCR tube, use pipette to mix DNA and primer by pipetting gently up and down; recap tubes.
6. Place PCR tubes in thermal cycler and program the cycler for:
   Initial Denature: 1 cycle at 94 °C for 2 min.
   Amplification: 40 cycles at 94 °C for 1 min (denature), 59 °C for 1 min (annealing), and 72 °C for 2 min (extension).
   Final Extension: 1 cycle at 72 °C for 10 min.
   Hold: 4 °C indefinitely.

3. 3% Agarose Gel Preparation

1. Using lab or masking tape, securely tape off the open ends of the gel tray. Ensure tape is sealed to the edges of the tray to prevent molten agarose from leaking out.
2. Weigh out 3 g of agarose into a 250-mL or larger Erlenmeyer flask.
3. Add 100 mL of 1x TAE buffer (purchased or prepared from concentrate).
4. Using a magnetic hot plate with a stir bar, heat the beaker until agarose is completely dissolved in the buffer and the solution is boiling and turns clear. Alternatively, a microwave oven can be used by placing beaker in oven and heating for 30 s intervals, using a stir rod to mix every 10 s, repeating until mixture is boiling and turns clear.
5. Invert a 50-mL Erlenmeyer flask into the opening of the 250-mL flask to act as a reflux, preventing agarose solution from evaporating.
6. Allow agarose solution to cool to 60 °C, and pour 30-50 mL into each taped gel tray.
7. Place a gel comb into the first pair of notches to create gel wells.
8. Allow gel to cool completely before removing tape and comb. Gel will solidify and turn from clear to cloudy when ready, approximately 10-20 min.

4. Electrophoresis of PCR Products

1. Place a pre-made 3% agarose gel onto a gel tray or use a gel tray that has been used to cast a poured 3% agarose gel.
2. Slide gel tray into the electrophoresis chamber with the wells closest to the cathode (black) end.
3. Pour 1x TAE buffer into the chamber, enough to have 2 mm of buffer above the top of the gel tray.
4. Obtain PCR tubes from the thermal cycler and place in microtube holder.
5. Using a fresh pipette tip each time, add 10 µL of purchased Orange G loading dye (LD) to each sample and mix well.
6. Load 20 µl of the molecular weight ruler and 20 µL of each sample into the gel in the order indicated (Table 2).
7. Run gel electrophoresis for 30 min at 100 V.
8. Remove gel tray from chamber and slide gel to remove from tray. Place gel in a staining tray.
9. Immerse gel in purchased DNA gel stain for 5 min, carefully shaking tray to help distribute the stain throughout the gel.
10. Transfer the gel into a washing container and rinse with tap water (40-55 °C) for approximately 10 s.
11. Destain by washing 3x in warm tap water for 6 min each with gentle shaking for best results. If necessary, continue destaining in warm water until the desired contrast is reached.

Tube number	Primer	Dna Sample
1	20 µL Plant primer (green)	20 µL Non-GMO food control DNA
2	20 µL GMO primer (red)	20 µL Non-GMO food control DNA
3	20 µL Plant primer (green)	20 µL Test food DNA
4	20 µL GMO primer (red)	20 µL Test food DNA
5	20 µL Plant primer (green)	20 µL GMO positive control DNA
6	20 µL GMO primer (red)	20 µL GMO positive control DNA

Table 1. List of the appropriate tube numbers, primers, and DNA samples.

Well 1	Sample 1 Non-GMO food control with plant primers 20 µL.
Well 2	Sample 2 Non-GMO food control with GMO primers 20 µL.
Well 3	Sample 3 Test food with plant primers 20 µL.
Well 4	Sample 4 Test food with GMO primers 20 µL.
Well 5	Sample 5 GMO positive DNA with plant primers 20 µL.
Well 6	Sample 6 GMO positive DNA with GMO primers 20 µL.
Well 7	PCR molecular weight ruler 20 µL.
Well 8	Leave empty.

Table 2. The appropriate order to load 20 µL of the molecular weight ruler and 20 µL of each sample into the gel.