TITLE: DNA Extraction - Qiagen DNeasy Blood and Tissue Kit UCC &amp; IMR eWHALE
AUTHORS: Lauren Rodriguez, Lorenzo De Bonis, James McKenna

[DESCRIPTION]
This DNA extraction protocol was used in Rodriguez et al., 2024 (in prep) for the extraction of environmental DNA from samples collected off of the coast of Ireland (Atlantic) and Norway (Norwegian Sea) as a part of the eWHALE project. Work was carried out by researchers at University College Cork (UCC) and the Institute for Marine Research (IMR). The protocol outlines lysis from Sterivex (0.45 µm pore size; Merck Millipore ID: SVHV010RS) eDNA filters and DNA extraction of lysates using the Qiagen DNeasy Blood and Tissue Kit. Mostly, the protocol follows the official version with spin columns published by Qiagen for the DNeasy Blood and Tissue Kit (available here: https://www.qiagen.com/us/resources/resourcedetail?id=68f29296-5a9f-40fa-8b3d-1c148d0b3030&lang=en), however, researchers at IMR used a QiaVAC 24 Plus vacuum system (ID: 19413, Qiagen) rather than centrifugation for spin column steps. 

[BEFORE_START]
Add the appropriate amount of ethanol (96-100%; required volume is listed on the label of each bottle) to Buffers AW1/AW2 if necessary (they are included in the kit as concentrates and need to be diluted). 

[GUIDELINES]
Lysis should be done in a laminar flow hood. 
Always change pipette tips while working with different lysates. 
All centrifugation steps should be carried out at room temperature (15–25°C). 
All reagents in the DNeasy extraction kit are viable for 1 year under the appropriate conditions (kept in a dry space at room temperature). 
If Buffer AL precipitates at any point, warm to 56°C until the precipitate disolves. 

[STEPS]
SECTION: Lysis
1. Remove filters from the freezer and allow to thaw at room temperature for 45 min . Ensure that all filter capsules are properly closed and labeled. 

SECTION: Lysis
2. Invert filters a couple of times to agitate lysate. 
SECTION: Lysis
3. Incubate filter capsules at 56 °C for 180 min . Invert filters every 30 minutes. 

SECTION: Lysis
4. Label 2 mL screw cap tubes or microcentrifuge tubes for all   . 

SECTION: Lysis
5. Using a 2 or 3 mL syringe, evacuate the lysis buffer from the filter capsule by pushing in 1 mL of air (while holding the filter horizontally) then evacuating the lysis buffer (while holding the filter vertically). 
SECTION: Lysis
6. Add the   from the syringe (0.5 - 2 mL) to the associated labeled tube. 

SECTION: Extraction
7. After incubation, vortex tubes for 15 s  then add 200 µL Buffer AL. 

SECTION: Extraction
8. Vortex again for 15 s then add 200 µL ethanol (96-100%). 

SECTION: Extraction
9. Pipette the mixture (including any precipitate) into a labeled DNeasy Mini spin column placed in a 2 mL collection tube. 
SECTION: Extraction
10. Centrifuge 6000 x g, 1 min 

SECTION: Extraction
11. Discard the flow-through + collection tube.
SECTION: Extraction
12. Place the spin column into a new 2 mL collection tube and pipette 500 µL of Buffer AW1 from the DNeasy kit into the column. 

SECTION: Extraction
13. Centrifuge 6000 x g, 1 min 

SECTION: Extraction
14. Discard the flow-through + collection tube. 
SECTION: Extraction
15. Place the spin column into a new 2 mL collection tube and pipette 500 µL of Buffer AW2 from the DNeasy kit into the column. 

SECTION: Extraction
16. Centrifuge 20000 x g, 3 min 

SECTION: Extraction
17. Discard the flow-through + collection tube. 
 

SECTION: Extraction
18. Remove the spin column carefully from the collection tube (do not allow it to come into contact with flow-through). 
SECTION: Extraction
19. Place the spin column into a clean 1.5 or 2 mL microcentrifuge tube and pipette 200 µL Buffer AE onto the spin column membrane. 

SECTION: Extraction
20. Incubate the samples at room temperature for 1 min .

SECTION: Extraction
21. Centrifuge 6000 x g, 1 min to elute

SECTION: Extraction
22. Repeat the previous 2 steps (incubate + centrifuge).