DNA Barcoding PCR Protocol

Abstract

What does this experiment test?

This protocol will allow you to identify a fungus, bird or mammal you have sampled and extracted DNA from.

In the same way as the barcode on an item you buy at the supermarket can be scanned to bring up the details of the item, all species can be identified from their unique DNA barcode. The DNA barcode is a sequence of DNA that varies sufficiently between different species for them to be taxonomically differentiated, but is flanked by sequences of DNA that are the same for all of these species for primers to bind to. Primers are short sequences of DNA that indicate which region of DNA is to be amplified in a PCR.

What are the genetics?

This project will produce a sequence of DNA, or DNA barcode, that can be sent for sequencing and compared against existing DNA databases to find the identity of the fungus, fish or mammal sampled.


Protocol

In this project, you will first extract DNA from a sample collected from a fungus, bird or mammal. This will take about 20 minutes. After this, you will use PCR to amplify the DNA barcode region that is relevant to your sample. This will take about 3 hours, but most of it will be waiting time. Finally, you will visualise the results using gel electrophoresis, which will take about 1 hour.
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 using the DNA Extraction for Barcoding or DNA Extraction from Feathers protocol. It will take ~20 minutes, 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 DNA barcode for your samples. The primer mix you use will depend whether you have sampled a fungus, bird or mammal:

    • ITS3/ITS4 for fungi
    • Bird F1/Bird R1 for birds
    • LCO1490/HCO2198 for mammals

    You will need the DNA template sample (1), an empty PCR tube (2), the primer mix for this project (3), the mastermix (4), and PCR grade water (5). The total final volume of your tube will be 20μL.

    First add the mastermix. Set your micropipette to 4μL.

    Using a fresh pipette tip, transfer 4μL of the master mix into the empty PCR tube. Then discard your tip.

    Next add the primer mix. Set your micropipette to 2μL. Using a fresh pipette tip, transfer 2μL of the primer mix into the PCR tube. Then discard your tip.

    Now add the DNA template. 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 and primer mix. Then discard your tip.

    Finally add PCR grade water to make the total volume up to 20μl. Set your micropipette to 10μl.

    Using a fresh pipette tip, transfer 10μl of PCR grade water into the PCR tube. Then discard your tip.

    Place your PCR tube in the thermocycler block.

    Set up the thermocycler with the following PCR program:

    (For help setting up a PCR on your Bento Lab visit the PCR Thermocycler User Manual.)

    PCR program for ITS3/ITS4 primer mix for fungi:

    • 2 mins at 95°C
    • 35 cycles made of 3 steps
      • 30 secs at 95°C
      • 30 secs at 55°C
      • 60 secs at 72°C
    • 10 mins at 72°C

    Total run-time = 135 mins

    PCR program for Bird F1/R1 primer mix:

    • 1 min at 94°C
    • 6 cycles made of 3 steps:
      • 60 secs at 94°C
      • 90 secs at 45°C (touchdown)
      • 90 secs at 72°C
    • 35 cycles made of 3 steps
      • 60 secs at 94°C
      • 90 secs at 55°C
      • 90 secs at 72°C
    • 5 mins at 72°C

    Total run-time = 239 mins

    PCR program for LCO1490/HCO2198 primer mix:

    • 2 mins at 95°C
    • 35 cycles made of 3 steps
      • 60 secs at 95°C
      • 60 secs at 40°C
      • 90 secs at 72°C
    • 7 mins at 72°C

    Total run-time = 204 mins

    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 1 hour.

  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

    Your gel should show the DNA ladder, a single clear band in the positive control provided with the primer mix and a single clear band in your sample(s). This means your sample is ready to send for sequencing.

    Trouble-shooting:

    DNA ladder + a clear single band in positive control + no band in your sample(s) = your DNA extraction(s) have not been successful –> repeat this step with a fresh sample.

    DNA ladder + no band in positive control + no band in your sample(s) = the PCR has not been successful –> repeat this step making sure you include all components of the mastermix.

    DNA ladder + multiple bands in positive control +/ multiple bands in your sample(s) = there is primer dimer or contamination in your PCR –> repeat this step using 5X HOT FIREPol Blend Master Mix Ready to Load and increase the duration of the first step in the PCR program (1 or 2 mins at 94oC or 95oC) to 15 minutes.

    After you have taken good photos of the gel for your documentation, you can dispose of the gel in your general waste bin.

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