DNA Extraction from Feathers – HotSHOT Kit

Reagents

Consumables

Equipment

Abstract

This protocol describes how to extract a DNA sample from freshly plucked bird feathers using a HotSHOT DNA Extraction Kit and Bento Lab.

Sample Preparation

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

  1. Obtaining sample

    The least invasive sources of DNA are small feathers plucked from a bird’s breast. There are videos on YouTube that will show you how to do this in a way that is least distressing to the bird. For this DNA extraction protocol to work, the feathers need to be freshly plucked and not moulted.

    When you have the feathers, use a new scalpel/razor blade on a clean surface (e.g. a cutting mat/non-scratch surface) to prepare two small sections from each feather of approximately 2 mm3 diameter.

    • One section will be from the tip of the feather 1, which should have skin cells attached from being recently plucked.
    • One section will be where the first feathers sprout from the calamus 2, as this area contains a DNA-rich blood clot (Horváth et al. 2005).
    Feather with two indicated cutting regions for sampling DNA.

    Place the feather sections in a PCR tube labelled on the top and side in permanent marker. To avoid sample cross-contamination, sterilise your scalpel/razor blade and cutting surface between feathers.

    Even if you only have one sample, it’s good practice to label the tube clearly with a unique identifier for the feather used. 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.

  1. Tissue lysis of sample

    Make sure the feather sections are 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. Due to PCR inhibitors in this buffer, you need to prepare a 1 in 10 dilution of your DNA extraction in PCR grade water.

    To do this, pipette 180 μl of PCR grade water into a fresh PCR tube and label it with your sample name and “1/10”. Then add 20 μl of your DNA extraction and mix by inverting the PCR tube several times.

    The avian sexing PCRs are more successful if you leave the DNA extractions and 1 in 10 dilutions in the freezer overnight. Remove them from the freezer ~1 hour prior to setting up the PCR so they are partially thawed, and do not invert or mix the tubes prior to adding the extractions to your PCR reaction mix.