How To Extract DNA and Set Up PCR Reactions (GMO detection example using food sample DNA)

Summary

This video guides viewers through the process of detecting genetically modified organisms (GMOs) in food using PCR. It covers how to extract DNA from food samples, including both GMO and non-GMO foods, and prepare them for PCR analysis. The procedure involves grinding the food samples, extracting DNA, and setting up PCR reactions with plant and GMO master mixes. Safety and contamination prevention measures, such as cleaning equipment thoroughly and handling samples carefully, are emphasized throughout. The process helps determine if grocery store foods contain GM crops.

Takeaways

• 😀 The video demonstrates how to detect genetically modified organisms (GMOs) in food using PCR.
• 😀 The procedure involves extracting template DNA from food samples purchased at the grocery store.
• 😀 For DNA extraction, label two screw cap tubes, one for testing and one for a non-GM control sample.
• 😀 A mortar and pestle is used to grind the food sample into a slurry with distilled water.
• 😀 The slurry must be mixed thoroughly to ensure it is smooth enough to pipette.
• 😀 After grinding, 50 microliters of the slurry is transferred to the test tube labeled 'test'.
• 😀 Ensure the mortar and pestle are cleaned between uses to avoid contamination, with soap, bleach, and distilled water.
• 😀 Incubate the test and non-GM control tubes at 95°C for five minutes, then centrifuge for five minutes.
• 😀 DNA samples can be stored at 4°C for up to a month, but should not be frozen.
• 😀 For PCR setup, prepare tubes with extracted DNA and use aerosol barrier tips to prevent contamination.
• 😀 Pipette 20 microliters of plant and GMO master mixes into corresponding PCR tubes for each DNA sample.

Q & A

What is the purpose of the PCR detection process described in the video?

The purpose of the PCR detection process is to determine whether foods purchased from the grocery store contain genetically modified organisms (GMOs) by extracting DNA and analyzing it.

What is the first step in preparing the food sample for PCR testing?

The first step is to label two screw cap tubes: one for the 'test' sample and one for the 'non-GM' sample. You then weigh the food sample and record its mass.

How do you prepare the food sample for extraction?

You grind the food sample in a mortar with pestle, adding 5 milliliters of distilled water for every gram of food, and continue grinding until a smooth slurry is formed.

What should be done after grinding the food sample?

After grinding, you transfer 50 microliters of the slurry into the labeled screw cap tube, recap the tube, and shake or vortex to mix.

Why is it important to clean the mortar and pestle thoroughly between samples?

Cleaning the mortar and pestle is crucial to prevent PCR contamination, as any residue could potentially affect the results of subsequent tests.

What temperature and duration should the screw cap tubes be incubated at after sample preparation?

The screw cap tubes should be incubated at 95°C for five minutes.

How should the screw cap tubes be treated after incubation?

After incubation, the screw cap tubes should be placed in a centrifuge and spun for five minutes at maximum speed. If using a mini centrifuge, it should be spun for 10 minutes.

What is the recommended storage method for the samples after centrifugation?

The samples can be stored at 4°C for up to a month, but they should not be frozen.

What is required to set up the PCR reaction?

To set up the PCR reaction, you will need tubes with extracted DNA from the non-GM food and the test sample, GMO positive control DNA, six PCR tubes, and a micropipette set to 20 microliters.

Why is it necessary to use aerosol barrier tips when handling PCR samples?

Aerosol barrier tips are necessary to prevent the transfer of aerosols from the pipette to the samples, which could cause contamination and affect the PCR results.

What is the importance of pipetting DNA from the upper supernatant rather than the pellet?

Pipetting DNA from the upper supernatant is important because the pellet contains the insta gene matrix, which can inhibit the PCR reaction if transferred.

What should be done after pipetting the DNA into each PCR tube?

After pipetting the DNA into each PCR tube, close the caps to prevent contamination and repeat the process for the other DNA samples.