DNA Extraction from Feathers

Sample Preparation

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

Obtaining sample

The least invasive sources of DNA are small feathers plucked from a bird’s breast. This extraction protocol will not work for moulted feathers.

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 feather approximately 2 mm³ diameter. For very small feathers include the base part of the feathers. For larger feathers cut a section very near the junction of the base and the first feather barbs – this area contains a DNA-rich blood clot (Horváth et al. 2005).

Place the sample in a PCR tube labelled on the top and side in permanent marker. To avoid sample cross-contamination, wash hands or change gloves between feathers and use a new scalpel/razor blade and 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 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.

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

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 95^oC 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.

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 Avian Sexing 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.