DNA Extraction - Qiagen DNeasy Blood and Tissue Kit UCC & IMR eWHALE
Lauren Rodriguez, Lorenzo De Bonis, James McKenna
Abstract
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).
Steps
Lysis
Remove filters from the freezer and allow to thaw at room temperature for 0h 45m 0s . Ensure that all filter capsules are properly closed and labeled.
Invert filters a couple of times to agitate lysate.
Incubate filter capsules at 56°C for 3h 0m 0s . Invert filters every 30 minutes.
Label 2 mL screw cap tubes or microcentrifuge tubes for all Sample .
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).
Add the Sample from the syringe (0.5 - 2 mL) to the associated labeled tube.
Extraction
After incubation, vortex tubes for 0h 0m 15s then add 200µL Buffer AL.
Vortex again for 0h 0m 15s then add 200µL ethanol (96-100%).
Pipette the mixture (including any precipitate) into a labeled DNeasy Mini spin column placed in a 2 mL collection tube.
Centrifuge 6000x g
Discard the flow-through + collection tube.
Place the spin column into a new 2 mL collection tube and pipette 500µL of Buffer AW1 from the DNeasy kit into the column.
Centrifuge 6000x g
Discard the flow-through + collection tube.
Place the spin column into a new 2 mL collection tube and pipette 500µL of Buffer AW2 from the DNeasy kit into the column.
Centrifuge 20000x g
Discard the flow-through + collection tube.
Remove the spin column carefully from the collection tube (do not allow it to come into contact with flow-through).
Place the spin column into a clean 1.5 or 2 mL microcentrifuge tube and pipette 200µL Buffer AE onto the spin column membrane.
Incubate the samples at room temperature for 0h 1m 0s .
Centrifuge 6000x g to elute
Repeat the previous 2 steps (incubate + centrifuge).
