Lab 9_ In-Lab _ Top Hat

Laboratory 9: Investigating Genetically Modified Food

Overview

  • Objective: Extract genomic DNA from food samples and analyze for genetically modified (GM) DNA sequences.

  • Procedure:

    • Week 1: DNA extraction and PCR amplification.

    • Week 2: Gel electrophoresis to detect and visualize DNA sequences.

Week 1: DNA Extraction

Part 1: Protocol for Extraction of DNA from Food Samples

  • Food Samples:

    • A control non-GM food (e.g., oats) and a test food (fresh corn, soybeans, processed items like cornmeal).

  • Extraction Process:

    1. Weigh food sample (1 g) and grind in water to create slurry.

    2. Mix slurry with InstaGene matrix in a screwcap tube.

    3. Incubate in water bath for 5 minutes to allow DNA release.

    4. Boiling denatures enzymes (DNases) that degrade DNA.

    5. Centrifuge to separate InstaGene matrix from DNA.

    6. Resulting supernatant contains extracted DNA to be used in PCR.

Safety Note

  • Always process non-GMO control before test sample to minimize contamination risks.

Week 1: Part 1 Steps

  1. Label screwcap tubes: "non-GMO" and "test".

  2. Measure and grind non-GMO control (1 g) with 5 mL distilled water.

  3. Add 50 µL of slurry to "non-GMO" tube with InstaGene matrix and mix.

  4. Clean mortar, then repeat for test food sample (1 g).

  5. Place both tubes in 95°C water bath for 5 minutes.

  6. Notify instructor for centrifuge processing.

Week 1: Part 2 - Preparing Samples for PCR

  • Create PCR reactions for DNA samples: Obtain two samples and a positive control for GM DNA.

  • First PCR Reaction:

    • Control to check if plant DNA was successfully extracted. Use Plant master mix (PMM) to amplify common chloroplast gene sequences using green primers.

  • Second PCR Reaction:

    • Check for presence of GM DNA using GMO master mix (GMM) with red primers, targeting common GM sequences.

PCR Tube Setup

  • Number PCR tubes from 1 to 6 with contents as follows (Refer to Figure 9.3):

    • 1: Non-GMO DNA + PMM (green)

    • 2: Non-GMO DNA + GMM (red)

    • 3: Test food DNA + PMM (green)

    • 4: Test food DNA + GMM (red)

    • 5: GMO positive control DNA + PMM (green)

    • 6: GMO positive control DNA + GMM (red)

Week 2: Gel Electrophoresis of PCR Products

  1. Set up electrophoresis apparatus.

  2. Add 10 μL of Orange G loading dye to each PCR tube, and mix.

  3. Prepare a 3% agarose gel using 1.5 g agarose in 50 mL 1X TAE buffer.

  4. Microwave agarose until dissolved; cool to ~60°C before adding Syber Safe dye and mixing.

  5. Pour gel into electrophoresis tank and allow to solidify.

  6. Load PCR samples, following load volume specifications:

    • Lane 1: Non-GMO control with plant primers

    • Lane 2: Non-GMO control with GMO primers

    • Lane 3: Test food with plant primers

    • Lane 4: Test food with GMO primers

    • Lane 5: GMO positive DNA with plant primers

    • Lane 6: GMO positive DNA with GMO primers

    • Lane 7: PCR molecular weight ruler

  7. Connect the gel chamber to power supply and run at 100 V for 30 minutes.

  8. After completion, notify instructor for results analysis and dispose of gel properly.