DNA Extraction for Barcoding

Reagents

Consumables

Equipment

Abstract

This protocol describes how to extract DNA from a fungus or mammal sample using Bento Lab.

Sample Preparation

In this step you will prepare your samples for DNA extraction.

  1. Obtaining sample

    Collect a sample from the fungus or mammal you would like to DNA barcode.

    • For macroscopic fungi (mushrooms), break off a small section from the edge – no need to take the entire fruiting body.
    • For microscopic fungi, use a sterile swab to collect mycelia from the culture.
    • For live mammals, obtain a spit or blood sample.
    • For meat products, use fresh or remove a chunk from a frozen sample and allow to defrost.

    Wear gloves whilst doing so to prevent contaminating the sample with human DNA.

    Use a new scalpel/razor blade on a clean surface (e.g. a clean piece of paper towel on top of cutting mat/non-scratch surface) to prepare a very small section of sample approximately 5 mm3 diameter. At this stage, if possible, try to shave off surfaces of the sample that have been exposed as these could be contaminated with microorganisms.

    Place the sample in a PCR tube labelled on the top and side in permanent marker. To avoid sample cross-contamination, sterilise or change gloves and scalpel/razor blade between samples and use a new piece of paper towel each time.

    Even if you only have one sample, it’s good practice to label the tube clearly with a unique identifier. It’s also a good idea to mark on the tube the date and to keep a note somewhere of which samples were prepared, and when.

DNA Extraction

In this step you will extract DNA from the samples prepared above.

Prior to starting this section of the protocol, remove the HotSHOT DNA Extraction Kit from storage at -20oC and allow both of the solutions inside to defrost.

  1. Tissue lysis of sample

    Make sure the sample is at the base of the tube before starting the DNA extraction; flick or tap the tube on a hard surface until this happens and then keep tube upright.

    Pipette 75 μl of Alkaline Lysis Solution (from the HotSHOT DNA Extraction Kit) into each labelled PCR tube containing a sample, using a fresh pipette tip each time. To do this, set the 20-200 μl adjustable pipette to 75 μl and use the 2-200 μl pipette tips.

    Wear gloves and be careful when handling the Alkaline Lysis Solution. If it comes into contact with skin wash the affected area immediately.

    Place the sealed PCR tubes in the thermocycler and use the heat block function to heat tubes at 95oC for 20 minutes.

    Select heat block function (2) and then set the temperature to 95oC for 20 minutes.

    Leave tubes to cool and then pipette 75 μl of HotSHOT Neutralising Buffer (from the HotSHOT DNA Extraction Kit) into each labelled PCR tube containing a sample, using a fresh pipette tip each time.

    Mix the solutions thoroughly by holding each PCR tube between your thumb and forefinger and inverting the tube several times.

  2. Labeling and storage

    The PCR tube now contains DNA in neutralised buffer. It is called the template sample, and can now be used in Step 2 of the DNA Barcoding Protocol.

    If you are not using the template sample in another protocol right away, store it in the freezer at around -20°C. This will preserve the sample.