TITLE: DNA extraction from feathers
AUTHORS: George  Olah

[DESCRIPTION]
This protocol describes a DNA extraction method from feathers collected non-invasively in the wild.

[GUIDELINES]
Set up the extraction on a clean lab bench in a PCR-free area. Always use a negative control during each batch of extractions.

[STEPS]
SECTION: Collection &amp; Preparation
1. When collecting in the field, avoid touching the tip of the feathers. If wet, dry the feather on paper towel before storing it.

Place each feather sample in a separate paper envelope, labelling: individual sample ID, collection date, location, species, collector's name, etc.

Store the envelopes with samples in an airtight dry-box with Silica gel crystals at Room temperature.
SECTION: Collection &amp; Preparation
2. For each feather sample, prepare and label an   with the unique sample ID.

SECTION: Collection &amp; Preparation
3. For each feather sample, get:
a clean A4 paper sheet
a pair of  
a sterile  .

SECTION: Isolation
4. Put on the  , remove the feather sample from the envelope and place it on the clean A4 paper sheet.
SECTION: Isolation
6. Remove a   from the sterile packaging.
Large feathers: carefully cut out a window around the blood clot from the superior umbilicus of the feather (this can usually be seen just below the vane).
See details in:
 
Small feathers: Chop up the entire shaft of small feathers (<20 mm).
 
  

SECTION: Isolation
7. Place the isolated sample into the corresponding Eppendorf tube with the correct sample ID.
Dispose the   to a yellow sharps container.

SECTION: Isolation
8. Put the remaining part of the feather back to its envelope.
Dispose the A4 paper and the gloves.

SECTION: Isolation
9.    and repeat the steps for each feather sample, avoiding cross-contamination.

SECTION: Isolation
5. Carefully clean the surface of the feather with   and a paper towel.

SECTION: Lysis
11. Add 180 µL of   to each tube.
SECTION: Lysis
12. Add 20 µL of   20 mg/mL to each tube.

SECTION: Lysis
14. Vortex each tube for 20 s.

SECTION: Lysis
15. Incubate 60 min at 56 °C on a block heater.

SECTION: Lysis
17. Incubate   at 56 °C on a block heater.
SECTION: Lysis
16. Vortex each tube for 20 s.

SECTION: Purification
18. Unpack appropriate number of spin column + collection tubes (according to the number of samples you are extracting) from   and label them with the sample IDs accordingly.

SECTION: Purification
20. Add 200 µL of   to each Eppendorf tube.

SECTION: Purification
19. Vortex each Eppendorf tube with the samples for 15 s.
Spin down briefly.

SECTION: Purification
21. Vortex for 15 s.
Spin down briefly.

SECTION: Purification
22. Incubate 45 min at 70 °C on a block heater.

SECTION: Lysis
13. Add 10 µL of   to dissolve Creatine.
SECTION: Purification
23. Spin down briefly and add 210 µL of   to each Eppendorf tube.

SECTION: Purification
24. Vortex and incubate for 5 min at Room temperature.

SECTION: Purification
25. Spin down briefly and pipette liquid from the Eppendorf tubes to the correspondingly labelled spin columns. Use   at this step.

SECTION: Purification
26. Centrifuge 10000 rpm, 1 min.

SECTION: Purification
27. Discard collection tube with the filtrate and place the spin column in a clean collection tube.
SECTION: Purification
28. Add 500 µL of   and centrifuge 10000 rpm, 1 min.

SECTION: Purification
29. Discard collection tube with the filtrate and place the spin column in a clean collection tube.
SECTION: Purification
30. Add 500 µL of   and centrifuge 12000 rpm, 3 min.

SECTION: Recovery of DNA
33. Discard collection tube with the filtrate and place spin column into a clean 1.5 mL Eppendorf tube (with lid cut off).
SECTION: Recovery of DNA
32. Prepare as many   as samples in your batch, and cut off their lids.

SECTION: Recovery of DNA
34. Add 100 µL of preheated   and incubate at 70 °C for 5 min on a block heater.
SECTION: Recovery of DNA
36. Centrifuge incubated samples 10000 rpm, 1 min.

SECTION: Recovery of DNA
37. Carefully lift the spin column, pipette up the eluted sample from the Eppendorf tube (~100 uL), place back to the spin column into the Eppendorf tube, and pipette the solution back to the center of the filter.

 

SECTION: Recovery of DNA
39. Centrifuge 10000 rpm, 1 min.

SECTION: Recovery of DNA
38. Incubate at 70 °C for 1 min.

SECTION: Recovery of DNA
40. Transfer filtrate to labeled  .

SECTION: Recovery of DNA
41. Store screw cap tubes in fridge at 4 °C for short-term or in a freezer at -20 °C for long-term storage.

SECTION: Isolation
10. Negative control: label an empty tube as negative control for the subsequent steps.

SECTION: Recovery of DNA
31. Preheat appropriate volume of   (100 µL x number of samples + a little extra) to 70 °C.
SECTION: Recovery of DNA
35. Prepare   and label them for final storage (species, sample ID, date of extraction, amount in μL).