Meat Detection

Beef vs Horse

Reagents

  • PCR Materials bag
    • PCR beads (1)
  • Horsemeat Primers (stored in the green refrigeration bag)
  • Gel electrophoresis
    • Agarose Tablet (1)
    • Safe DNA Stain
    • Loading Dye
    • 100bp DNA Ladder
    • Prepared 0.5x TBE buffer (prepared in Introduction to Gel Electrophoresis)
  • Salt. Not included.

Consumables

  • Transfer Pipette (1)
  • Pipette Tips (7)
  • Microcentrifuge Tubes (1)
  • PCR tubes (2)

Equipment

  • Shot glass or small glass. Not included.  
  • Micropipette
  • Bento Lab
  • Microwave

Abstract

In this project, we are conducting species identification, to determine whether a meat samples contains beef or horse meat. To do this, we will examining the Cytochrome b gene, which is found in the mitochondria of all species. Mitochondrial DNA is commonly used for species identification because the mitochondrial DNA remains more intact than genomic DNA when the food is processed.

What are we testing?

The Cytochrome b gene varies slightly across species, including in size. This experiment takes advantage of that feature, as we will amplify the same genes for both species, but the DNA fragment produced will be of different sizes, which allow us to infer what species are present.

Overview

What are the possible results for this experiment?

The Cytochrome b gene is specific to different species. With the primers in this experiment we can determine the presence of the Cytochrome b gene for beef, and the Cytochrome b gene for horses.

There are two possible bands. If the sample is pure beef, we will only observe one DNA band for the bovine gene. If there is presence of horse meat, we will see a DNA band for the equine gene.

  • Bovine: the bovine Cytochrome b gene has been successfully copied, there is beef in the sample, and the PCR reaction has worked.
  • Equine: the equine Cytochrome b gene has been successfully copied, which suggests  there is presence of horsemeat in the sample.

Protocol

In this project, you will first extract DNA from tissue samples. This will take about 20 minutes. After this, you will use PCR to amplify two variations of the Cytochrome b gene. This will take about 90 min, but most of it will be waiting time.
Finally, you will visualise the results using Gel Electrophoresis, which will take about 45 min.
At the end of each section, you can continue right away, or store your samples and continue later.

  1. DNA Extraction

    First, obtain the DNA sample. Use the DNA Extraction from tissue. It will take ca 20 min, at the end of which you should have a clean DNA template sample in a PCR tube.

  2. PCR

    In this step, you will use PCR to amplify the Cytochrome b gene.

    The experiment uses a version of DNA barcoding, where the same target DNA sequence across different species is examined to identify the species present.

    You will need the DNA template sample (1), a PCR tube with a mastermix bead (2) and the primer mix for this project (3).

    Set your micropipette to 20μL.

    Using a fresh pipette tip, transfer 20μL of the primer mix into the PCR tube with the mastermix bead. Then discard your tip.

    Now set your micropipette to 4μl.

    Using a fresh pipette tip, transfer 4μl of the DNA template sample from the sample tube into the PCR tube with the mastermix bead and primer mix. Then discard your tip.


    Place your PCR tube in the thermocycler block.

    Set up the thermocycler with the following PCR program:

    • 120 sec at 94°C
    • 35 cycles made of 3 steps
      • 30 sec at 94°C
      • 30 sec at 60°C
      • 30 sec at 72°C
    • 120 sec at 72°C

    If you need help operating the Bento Lab thermocycler, check the manual. You can use the PCR  preset (1), then modify (2) the program to the required settings (3) before running the program (4).

    The program will run for ca 2 hours. When it is finished, you can keep the result in the freezer, or use it right away for gel electrophoresis.

  3. Gel Electrophoresis

    Follow the Gel Electrophoresis Protocol to cast a gel and run it with your PCR result, and a 100bp ladder. This should take about 40 min.

  4. Visualising the Gel

    After the gel run has completed, you can visualise your results.

    Continue to wear gloves as you handle the gel

    Open the orange lid of the gel box, and wipe off the condensation.  

    Gently pour out the buffer, and dispose of the buffer down a drain.

    Drain disposal of TBE running buffers is a standard waste disposal procedure followed by research labs. If you have questions, get in touch with us.

    Place the gel box onto the Bento Lab transilluminator surface. In order to get best visibility, you should do this in a room as dark as possible.

    Turn Bento Lab on, select the Gel Electrophoresis module, and turn on the Transilluminator light.

    Hold the orange filter lid over the gel to visualise the DNA bands. For documentation, use your mobile phone to take a clear picture of the gel. Rather than holding the lid over the gel, you can hold the lid directly in front of your camera lense.

    If the bands are faint, try to reduce the light in the room, e.g. by closing the curtains and turning off the lights.  You can also carefully take the gel out of the gel box and place it directly onto the transilluminator. Wear gloves when doing this, and be careful not to break the gel.

  5. Analysing your results

    Compare the picture of your gel to this example result, which has been run with all variations. Your sample should correspond to one of these variations.

    1 – Ladder – 100 bp DNA Ladder

    2 – Beef
    Cytochrome b, beef variation (274 bp)
    This result shows presence of the bovine gene, which confirms that our sample includes beef. There is no DNA band for the equine genus.

    3 – Horse
    Cytochrome b, horse variation (439 bp)
    This result shows the presence of the equine gene. This suggests your sample is 100% pure horse meat!

    4 – Beef and Horse
    Both Cytochrome b variations: Beef (274 bp), Horse (439 bp)
    This result shows both the presence of the bovine gene and the equine gene. This suggests it’s possible there is both horse and beef in our meat sample!

    After you have taken good photos of the gel for your documentation, you can dispose of the gel in your regular trash.

    Disposal of agarose gels is a standard waste disposal procedure followed by research labs. If you have questions, get in touch with us.