TITLE: DNA extraction protocol for historical toe pad samples from birds
AUTHORS: Dave Lutgen, Reto Burri

[DESCRIPTION]
Museums hold collections of specimens from vast taxonomic and geographic ranges that constitute rich resources for research into the origins, evolution, and maintenance of biodiversity on earth. However, specimens are often not prepared for specific research purposes. Especially the isolation of adequate quantities of DNA for genomic research can be challenging. Here, we report a modified protocol of a commercially available kit optimized to reliably extract adequate quantities of DNA from bird toepads and dried skin from sutures of museum specimens for whole-genome resequencing.

[STEPS]
SECTION: Tissue preparation
3. Put toe pad/breast skin tissue (2-3mg) into   (use scalpel tip to transfer). 

SECTION: Tissue preparation
5. Mince the tissue with a pointed scalpel blade. Use a new blade for each sample!


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

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

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

SECTION: Digestion
9. Mix well by vortexing. Incubate tubes at  . Make sure the timer is on infinity.

SECTION: Digestion
10. After 360 min, centrifuge the tubes and add 20 µL of  to the remaining tissue. Use the pestle to further crush tissue parts. Pipet a second volume of 180 µL  over the pestle to ensure all tissue remains in the digestion.

SECTION: Pre-extraction preparations
12. Preheat  at 56 °C. This step prevents the formation of precipitates when  is added to the digestion.

SECTION: Pre-extraction preparations
13. Prepare a 1:1 dilution of and  . Preheat at 37 °C.

SECTION: Pre-extraction preparations
14. Prepare and label  for each extraction (2 for 2 elutions)

SECTION: Preparation
1. This protocol uses the  with 2x the amount/volumes of 
 
 
  
 .
SECTION: Preparation
2.  Prepare a   per sample.
 Prepare a   to store the micropestle separately for each sample.
 Preheat  at 56 °C. Adding preheated  prevents the formation of potential precipitates.

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

SECTION: Digestion
16. Add 4 µL  . Mix by vortexing, and incubate at Room temperature for 10 min.

SECTION: Digestion
17. Vortex for 15 s. Add 400 µL of preheated 56 °C  . Mix very quickly. A coagulate will form. Dissolve this in the heat block at   

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

SECTION: Extraction
20. Apply 650 µL of the lysate to the column. Centrifuge at 8000 rpm, 1 min . Discard flow-through, dry the collection tube, and place the column back into the collection tube.

SECTION: Extraction
21. Apply the rest of the lysate. Centrifuge at8000 rpm, 1 min. Discard flow-through and collection tube.

SECTION: Extraction
22. Place the column into a new collection tube. Add 500 µL  . Centrifuge at 8000 rpm, 1 min . Discard flow-through and collection tube.

SECTION: Extraction
23. Place the column into a new collection tube. Add 500 µL  . Centrifuge at 8000 rpm, 1 min . Discard flow-through, dry collection tube, and place column back into it.

SECTION: Extraction
24. Centrifuge at 14000 rpm, 2 min to dry the membrane. Make sure no ethanol is left anywhere in or on the column. Discard the collection tube.

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

SECTION: Extraction
26. Put the column into the thermoshaker    Then spin at 8000 rpm, 2 min .

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

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

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

SECTION: Digestion
15.1. 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)
SECTION: Tissue preparation
4. Museum toe pad samples might have been treated with chemicals to better conserve them, therefore, we will rince the tissue before starting the digestion
SECTION: Tissue preparation
4.1. Add 1000 µL of  to wash down chemicals on the toe pad. Incubate at    Discard all . Make sure no is left.
SECTION: Results
28.