DNA Extraction - Qiagen BioSprint® 96 Workstation using the Biosprint 96 tissue protocol UIBK eWHALE
Lauren Rodriguez, Bettina Thalinger
Abstract
This DNA extraction protocol is used in Rodriguez et al., 2024 (in prep) for the extraction of environmental DNA from samples collected in the North-East Atlantic and Mediterranean Sea as a part of the eWHALE project. The protocol outlines lysis from both Sylphium (https://sylphium.com/eng/) and Smith-Root (https://www.smith-root.com/) eDNA filters and DNA extraction of lysates using the Qiagen BioSprint® 96 Workstation automated extraction robot + BioSprint 96 DNA blood Kit (ID: 940057). For further information on this protocol, please refer to Qiagen (2012) BioSprint® 96 DNA Handbook. Available via Qiagen: https://www.qiagen.com/us/resources/resourcedetail?id=64902c5d-9c3c-4fe3-a3f7-668c4704d9eb&lang=en
Before start
Sample collection and preparation:
Seawater was collected in the waters surrounding the Azores Islands as well as in the Ligurian Sea filtered using Sylphium filter capsules with 0.45µm filters. All filters were stored at -20 °C until being shipped or transported to the laboratory at the University of Innsbruck (Austria). Once arriving at the laboratory, filters were again stored at -20 °C.
Lab work:
Clean all workbenches with bleach and ethanol prior to use.
Thaw all samples.
Steps
Lyse sample
Smith-Root Filters
Label 2 mL tube for each Sample (+ control)
14000rpmCentrifuge tubes with the 2 mL lid open.
Throw away the filters from each tube - lysate for extraction remains at the bottom of the 2 mL tube.
Remove filter housing from the plastic bag. The yellow half of the filter should be facing the bottom of the bag. Use an Erlenmeyer flask to hold the filter upright while the filter is being opened.
Discard the beige filter top.
Using two pairs of forceps and a pair of scissors, all which have been sterilized by flame 3x each, fold the "top" filter paper from each Sample into a triangle and put it into the corresponding 2 mL reaction tube (tip is at the bottom end).
Pipette 380µL TES buffer (an alternative to Qiagen DNeasy lysis buffer) and 20µL Proteinase K (total lysis buffer mixture = 400µL) to all 2 mL reaction tubes, taking care to saturate the filter paper.
Vortex filters/turn them upside down a few times.
Incubate all 2 mL tubes at 56°C for 3h 0m 0s
Remove from incubator, ,0h 0m 0s briefly to spin down lysate.
Transfer filter into a plastic inlet* and place the inlet + filter back into the original 2 mL tube in which the filter came from.
Sylphium Filters
Make sure that all filter capsules are labelled and closed properly on the top and bottom.
Incubate filters at 56°C for 3h 0m 0s
Label 2 mL for each Sample (+ control)
Use 3 mL to 10 mL syringes (which fit to Luer Locks) to evacuate the lysis buffer from each capsule. Hold the filter vertically, attach syringe to the bottom cap, unscrew the top cap then suck the lysate out.
DNA Extraction using Qiagen BioSprint® Workstation
Prepare extraction reagents according.
Pipette 650µL of AW1 Buffer into each well of Wash Plate 1 (S-Block).
Pipette 500µL of AW1 Buffer into each well of Wash Plate 2 (S-Block).
Pipette 500µL AW2 Buffer into each well of Wash Plate 3 (S-Block).
Pipette 500µL AW2 Buffer into each well of Wash Plate 4 (S-Block).
Pipette 500µL RNase free water (MilliQ water) + Tween 20 (Final concentration of 0.02%) to each well of Wash Plate 5 (Microplate).
Pipette 100µL Buffer TE or AE into each well of the Elution Plate (Microplate).
Prepare lysate bind plates (for DNA uptake).
Pipette 300µL AL Buffer and 300µL Isopropanol to each well in each bind plate.
Add 30µL MagAttract magnetic particles to each well in the FIRST bind plate.
Add 200µL lysate to each of the bind plates. Fill up any missing lysate volume (i.e., not up to 200µL ) with TES buffer (or DNeasy Lysis Buffer).
Place a new Rod Cover into the first bind plate. Start the appropriate program on the BioSprint ("DNA uptake") according to the number of bind plates (2-7 are available).
Using the last bind plate with DNA + Rod Cover as well as the other plates with all reagents, start the "eDNA extract" program on the BioSprint.
Pipette processed extracts (in the elution plate!) into Eppendorf tubes®
Cleanup
Carefully dispose of toxic BIoSprint waste into appropriate receptacle.
Wash all blocks/plates several times with water and subsequently incubate in a bleach-water bath for 1 day.
After 1 day, wash the blocks/plates in the dishwasher for reuse.
