Dec 06, 2021
DNA extraction from feathers
- 1Australian National University
- EBL_ANU
External link: https://doi.org/10.1007/s12686-016-0573-4
Protocol Citation: George Olah 2021. DNA extraction from feathers. protocols.io https://dx.doi.org/10.17504/protocols.io.bzu2p6ye
Manuscript citation:
Olah G, Heinsohn RG, Brightsmith DJ, Espinoza JR, Peakall R (2016) Validation of non-invasive genetic tagging in two large macaw species (Ara macao and A. chloropterus) of the Peruvian Amazon. Conservation Genetics Resources 8(4):499–509. doi:10.1007/s12686-016-0573-4
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 06, 2021
Last Modified: December 06, 2021
Protocol Integer ID: 54906
Keywords: birds, feathers, non-invasive, DNA, extraction
Abstract
This protocol describes a DNA extraction method from feathers collected non-invasively in the wild.
Image Attribution
Screenshot from the documentary "The Macaw Kingdom" (https://youtu.be/3ieppWouPxk).
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.
Materials
Gloves
70% Ethanol
100% Ethanol
scalpel blades
Proteinase K, 2mLQiagenCatalog #19131
DTT (1,4-Dithiothreitol)Sigma AldrichCatalog #10708984001
Eppendorf Safe-Lock Tubes 1.5 mL PCR clean colorless 500 tubesEppendorfCatalog #022363212
1.5 mL Eppendorf tubes
Microtubes 1.5ml with screw caps
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Protocol materials
1.5 mL Eppendorf tubes
Microtubes 1.5ml with screw caps
Gloves
70% Ethanol
100% Ethanol
scalpel blades
Proteinase K, 2mLQiagenCatalog #19131
Eppendorf Safe-Lock Tubes 1.5 mL PCR clean colorless 500 tubesEppendorfCatalog #022363212
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
DTT (1,4-Dithiothreitol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #10708984001
Eppendorf Safe-Lock Tubes 1.5 mL PCR clean colorless 500 tubesEppendorfCatalog #022363212
Gloves
scalpel blades
Gloves
scalpel blades
scalpel blades
70% Ethanol
Buffer ATL (tissue lysis buffer)QiagenCatalog #19076
Proteinase K, 2mLQiagenCatalog #19131
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Buffer AL, Lysis bufferQiagenCatalog #19076
DTT (1,4-Dithiothreitol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #10708984001
100% Ethanol
1 ml pipette tips
Buffer AW1QiagenCatalog #19081
Buffer AW2QiagenCatalog #19072
1.5 mL Eppendorf tubes
Buffer AEQiagenCatalog #19077
Microtubes 1.5ml with screw caps
Buffer AEQiagenCatalog #19077
Microtubes 1.5ml with screw caps
Safety warnings
Be careful when cutting feathers with the surgical blades, as the shafts of larger feathers are very hard and blades can slip. Aim to cut through with the pointy tip of the blades first.
Collection & Preparation
Collection & Preparation
30m
30m
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 .
15m
For each feather sample, prepare and label an Eppendorf Safe-Lock Tubes 1.5 mL PCR clean colorless 500 tubesEppendorfCatalog #022363212 with the unique sample ID.
15m
For each feather sample, get:
- a clean A4 paper sheet
- a pair of Gloves
- a sterile scalpel blades .
1m
Isolation
Isolation
1h
1h
Put on the Gloves , remove the feather sample from the envelope and place it on the clean A4 paper sheet.
1m
Carefully clean the surface of the feather with 70% Ethanol Contributed by users and a paper towel.
1m
Remove a scalpel blades 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:
CITATION
Small feathers: Chop up the entire shaft of small feathers (<20 mm).
Dry blood clot from the superior umbilicus of a large macaw feather.
2m
Place the isolated sample into the corresponding Eppendorf tube with the correct sample ID.
Dispose the scalpel blades to a yellow sharps container.
1m
Put the remaining part of the feather back to its envelope.
Dispose the A4 paper and the gloves.
1m
go to step #4 and repeat the steps for each feather sample, avoiding cross-contamination.
Negative control: label an empty tube as negative control for the subsequent steps.
1m
Lysis
Lysis
1d
1d
Add 180 µL of Buffer ATL (tissue lysis buffer)QiagenCatalog #19076 to each tube.
1m
Add 20 µL of Proteinase K, 2mLQiagenCatalog #19131 20 mg/mL to each tube.
1m
Add 10 µL 1M of DTT (1,4-Dithiothreitol)Sigma AldrichCatalog #10708984001 to dissolve Creatine.
1m
Vortex each tube for 00:00:20 .
20s
Incubate 01:00:00 at 56 °C on a block heater.
1h
Vortex each tube for 00:00:20 .
20s
Incubate Overnight at 56 °C on a block heater.
15h
Purification
Purification
2h
2h
Unpack appropriate number of spin column + collection tubes (according to the number of samples you are extracting) from QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504 and label them with the sample IDs accordingly.
10m
Vortex each Eppendorf tube with the samples for 00:00:15 .
Spin down briefly.
1m
Add 200 µL of Buffer AL, Lysis bufferQiagenCatalog #19076 to each Eppendorf tube.
1m
Vortex for 00:00:15 .
Spin down briefly.
1m
Incubate 00:45:00 at 70 °C on a block heater.
45m
Spin down briefly and add 210 µL of 100% EthanolContributed by users to each Eppendorf tube.
1m
Vortex and incubate for 00:05:00 at Room temperature .
5m
Spin down briefly and pipette liquid from the Eppendorf tubes to the correspondingly labelled spin columns. Use 1 ml pipette tips at this step.
2m
Centrifuge 10000 rpm, 00:01:00 .
1m
Discard collection tube with the filtrate and place the spin column in a clean collection tube.
1m
Add 500 µL of Buffer AW1QiagenCatalog #19081 and centrifuge 10000 rpm, 00:01:00 .
2m
Discard collection tube with the filtrate and place the spin column in a clean collection tube.
1m
Add 500 µL of Buffer AW2QiagenCatalog #19072 and centrifuge 12000 rpm, 00:03:00 .
4m
Recovery of DNA
Recovery of DNA
30m
30m
Preheat appropriate volume of Buffer AEQiagenCatalog #19077 (100 µL x number of samples + a little extra) to 70 °C .
3m
Prepare as many 1.5 mL Eppendorf tubes as samples in your batch, and cut off their lids.
3m
Discard collection tube with the filtrate and place spin column into a clean 1.5 mL Eppendorf tube (with lid cut off).
1m
Add 100 µL of preheated Buffer AEQiagenCatalog #19077 and incubate at 70 °C for 00:05:00 on a block heater.
5m
Prepare Microtubes 1.5ml with screw capsContributed by users and label them for final storage (species, sample ID, date of extraction, amount in μL).
5m
Centrifuge incubated samples 10000 rpm, 00:01:00 .
1m
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.
Note
This helps to increase the concentration of DNA in the extraction.
2m
Incubate at 70 °C for 00:01:00 .
1m
Centrifuge 10000 rpm, 00:01:00 .
1m
Transfer filtrate to labeled Microtubes 1.5ml with screw capsContributed by users .
1m
Store screw cap tubes in fridge at 4 °C for short-term or in a freezer at -20 °C for long-term storage.
Citations
Step 6
Horváth, M.B.; Martínez-Cruz, B.; Negro, J.J.; Kalmár, L.; Godoy, J.A.. An overlooked DNA source for non-invasive genetic analysis in birds
http://dx.doi.org/10.1111/j.0908-8857.2005.03370.x