TITLE: DNA Extraction - Qiagen BioSprint® 96 Workstation using the Biosprint 96 tissue protocol UIBK eWHALE
AUTHORS: Lauren Rodriguez, Bettina Thalinger

[DESCRIPTION]
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]
SECTION: Lyse sample
1. Smith-Root Filters
SECTION: Lyse sample
1.1. Label 2 mL tube for each    (+ control)
SECTION: Lyse sample
1.2. 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. 
SECTION: Lyse sample
1.3. Discard the beige filter top. 
SECTION: Lyse sample
1.4. 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    into a triangle and put it into the corresponding 2 mL reaction tube (tip is at the bottom end). 

SECTION: Lyse sample
1.5. Pipette 380 µL TES buffer (an alternative to Qiagen DNeasy lysis buffer) and 20 µL Proteinase K (total lysis buffer mixture = 400 µL) to all 2 mL reaction tubes, taking care to saturate the filter paper.
 

 



SECTION: Lyse sample
1.6. Vortex filters/turn them upside down a few times. 

SECTION: Lyse sample
1.7. Incubate all 2 mL tubes at 56 °C for 180 min 

SECTION: Lyse sample
2. Sylphium Filters
SECTION: Lyse sample
1.8. Remove from incubator,   briefly to spin down lysate. 

SECTION: Lyse sample
1.9. Transfer filter into a plastic inlet* and place the inlet + filter back into the original 2 mL tube in which the filter came from. 
 

SECTION: Lyse sample
1.10. 14000 rpm, 10 minCentrifuge tubes with the 2 mL lid open.  

SECTION: Lyse sample
1.11. Throw away the filters from each tube - lysate for extraction remains at the bottom of the 2 mL tube. 
SECTION: Lyse sample
2.1. Make sure that all filter capsules are labelled and closed properly on the top and bottom. 
SECTION: Lyse sample
2.2. Incubate filters at 56 °C for 180 min 

SECTION: Lyse sample
2.4. 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. 
 

SECTION: Lyse sample
2.3. Label 2 mL for each   (+ control)

SECTION: DNA Extraction using Qiagen BioSprint® Workstation
3. Prepare extraction reagents according. 
SECTION: DNA Extraction using Qiagen BioSprint® Workstation
3.1. Pipette 650 µL of AW1 Buffer into each well of Wash Plate 1 (S-Block). 

SECTION: DNA Extraction using Qiagen BioSprint® Workstation
3.2. Pipette 500 µL of AW1 Buffer into each well of Wash Plate 2 (S-Block). 
SECTION: DNA Extraction using Qiagen BioSprint® Workstation
5. Prepare lysate bind plates (for DNA uptake).
 

SECTION: DNA Extraction using Qiagen BioSprint® Workstation
3.3. Pipette 500 µL AW2 Buffer into each well of Wash Plate 3 (S-Block). 

SECTION: DNA Extraction using Qiagen BioSprint® Workstation
3.4. Pipette 500 µL AW2 Buffer into each well of Wash Plate 4 (S-Block). 

SECTION: DNA Extraction using Qiagen BioSprint® Workstation
3.5. Pipette 500 µL RNase free water (MilliQ water) + Tween 20 (Final concentration of 0.02%) to each well of Wash Plate 5 (Microplate). 

SECTION: DNA Extraction using Qiagen BioSprint® Workstation
5.1. Pipette 300 µL AL Buffer and 300 µL Isopropanol to each well in each bind plate. 

SECTION: DNA Extraction using Qiagen BioSprint® Workstation
5.2. Add 30 µL MagAttract magnetic particles to each well in the FIRST bind plate. 
 


SECTION: DNA Extraction using Qiagen BioSprint® Workstation
5.3. Add 200 µL lysate to each of the bind plates. Fill up any missing lysate volume (i.e., not up to 200 µL ) with TES buffer (or DNeasy Lysis Buffer). 

SECTION: DNA Extraction using Qiagen BioSprint® Workstation
6. 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). 
 

SECTION: DNA Extraction using Qiagen BioSprint® Workstation
7. 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. 
SECTION: DNA Extraction using Qiagen BioSprint® Workstation
8. Pipette processed extracts (in the elution plate!) into Eppendorf tubes®
SECTION: Cleanup
9. Carefully dispose of toxic BIoSprint waste into appropriate receptacle.
SECTION: Cleanup
10. Wash all blocks/plates several times with water and subsequently incubate in a bleach-water bath for 1 day. 
SECTION: Cleanup
11. After 1 day, wash the blocks/plates in the dishwasher for reuse. 
SECTION: DNA Extraction using Qiagen BioSprint® Workstation
4. Pipette 100 µL Buffer TE or AE into each well of the Elution Plate (Microplate).