DNA extraction protocol for historical toe pad samples from birds

This protocol uses the with 2x the amount/volumes of

Prepare a per sample.

Prepare a to store the micropestle separately for each sample.

Prehet a56°C . Adding preheate prevents the formation of potential precipitates.

Put toe pad/breast skin tissue (2-3mg) into (use scalpel tip to transfer).

Museum toe pad samples might have been treated with chemicals to better conserve them, therefore, we will rince the tissue before starting the digestion

Add 1000µL of to wash down chemicals on the toe pad. Incubate at 1000rpm Discard all. Make sure nois left.

Mince the tissue with a pointed scalpel blade. Use a new blade for each sample!

To make sure no tissue is left on the blade, pipet 80µL of over the scalpel blade, then remove it and ensure all tissue remains in the digestion.

Add 20µL of and start crushing the tissue with a micropestle. Keep the pestle in the tube.

Pipet 100µL of over the micropestle to ensure all tissue remains in the digestion. Transfer the pestle into the prepared for storage.

Mix well by vortexing. Incubate tubes at 900rpm. Make sure the timer is on infinity.

After 6h 0m 0s, centrifuge the tubes and add 20µL of to the remaining tissue. Use the pestle to further crush tissue parts. Pipet a second volume of 180µL over the pestle to ensure all tissue remains in the digestion.

Vortex well. Make sure all pieces of tissue are in solution and continue digestion overnight at 56°C. Leave to digest 900rpm . (Note: Tests with extraction after 24 and 40 hours show that DNA amount doubles within the additional 16 hours).

Preheat at 56°C. This step prevents the formation of precipitates when is added to the digestion.

Prepare a 1:1 dilution ofand . Preheat at 37°C.

Prepare and label for each extraction (2 for 2 elutions)

Once all tissue is properly digested, in case there are undigested parts (bones, feathers, etc), centrifuge the lysate at max speed for 0h 5m 0s, and transfer the lysate into a new tube. Else, continue with the next step.

Note: it is possible that with increased digestion time bigger pieces of tissue are becoming visible. This is likely due to increasing hydration/swelling of tissue pieces with prolonged digestion, and exactly what you want)

Add 4µL . Mix by vortexing, and incubate at Room temperature for 0h 10m 0s.

Vortex for 0h 0m 15s. Add 400µL of preheated 56°C . Mix very quickly. A coagulate will form. Dissolve this in the heat block at 900rpm

Turn temperature on the heat block down to 37°C. Preheat 1:1 diluted Buffer AE at 37°C.

Add 400µL ice-cold (-20°C) . Mix very well by vortexing. Incubate at room temperature for 10 min.

Apply 650µL of the lysate to the column. Centrifuge at 8000rpm . Discard flow-through, dry the collection tube, and place the column back into the collection tube.

Apply the rest of the lysate. Centrifuge at8000rpm. Discard flow-through and collection tube.

Place the column into a new collection tube. Add 500µL . Centrifuge at 8000rpm . Discard flow-through and collection tube.

Place the column into a new collection tube. Add 500µL . Centrifuge at 8000rpm . Discard flow-through, dry collection tube, and place column back into it.

Centrifuge at 14000rpm to dry the membrane. Make sure no ethanol is left anywhere in or on the column. Discard the collection tube.

Place the column into a labeled . Apply 20µL of preheated (37°C) 1:1 diluted to the column.

Put the column into the thermoshaker 0rpm Then spin at 8000rpm .

If you expect high(er) amounts of DNA, repeat steps and with another 20µLof 1:1 diluted .