TITLE: Automated eDNA Extraction from Estuarine Samples Using Magnetic Beads
AUTHORS: Fouad El Baidouri , Muriel Kelly, Heather L. Gilbert, Alison Watts

[DESCRIPTION]
This method presents an optimized protocol for the automated extraction of environmental DNA (eDNA) from estuarine samples using magnetic beads. The addition of Proteinase K to the lysis solution facilitates DNA solubilization at 56°C and mechanical rotation ensures thorough lysis. The DNA is then purified using a double ethanol wash and magnetic bead-based extraction on a KingFisher system. The method is designed for filters preserved in Longmire's buffer.  If a different preservation is used the first step should be modified.

[BEFORE_START]
Longmire’s buffer preparation: ensure that Longmire's buffer is prepared using PCR grade water.

[GUIDELINES]
Follow Personal Protective Equipment (PPE) procedures.
Clean workspace:  Thoroughly clean all work surfaces including the benchtop and equipment exteriors using a 5%-10% bleach solution followed by a distilled water rinse. Wipe down pipettes, pipette tip boxes, sharpies, pens and any other tools that will be used during the protocol to avoid cross contamination.
Incubator check: Verify that the incubator is operational and set to the correct temperature before use (56°C ).
Handling: Use aseptic techniques to avoid introducing contaminants into sample preparations. Include extraction blanks to check for cross contamination from adjacent wells.
Storage: If immediate DNA extraction is not possible, samples can be stored in Longmire's buffer at room temperature for up to 50 days (minimize exposure to light), or frozen at -20°C or -80°C for long-term storage.
Notes
Illumina Tune beads (CAT: 20060057) performed better than Ampure XP and are cheaper.

[STEPS]
SECTION: Sample Preparation
1. Perform the following steps in a biosafety cabinet under aseptic conditions
For samples collected on filters stored in Longmire's buffer (wet filters)
Pipette out all of the Longmire's buffer from the sample tube into a separate Eppendorf tube (leave the sample filter in the sample tube)
Pipette 490 μl per filter of the original Longmire's buffer back into the sample tube
Add 10 μl of Proteinase K per filter into the sample tube (total volume 500 μl)
Using the pipette tip, mix the reagents thoroughly
Using the same pipette tip, ensure the filter(s) are completely submerged in the Longmire's buffer/Proteinase K solution
 For dry filters, add 1000 μl of Longmire's solution to the tube, ensuring the filter is completely    submerged - Incubate for 90 min to overnight.
Pipette out all of the Longmire's buffer from the sample tube into a separate Eppendorf tube (leave the sample filter in the sample tube)
Pipette 490 μl per filter of the original Longmire's buffer back into the sample tube
Add 10 μl of Proteinase K per filter into the sample tube (total volume 500 μl)
Using the pipette tip, mix the reagents thoroughly
Using the same pipette tip, ensure the filter(s) are completely submerged in the Longmire's buffer/Proteinase K solution
5. After the program ends, immediately proceed with the Zymo cleanup steps using the elution plate, or store the plate at -20°C for future use. Dispose of chemical reagents according to Lab Hazardous Waste Management Plan.
SECTION: Sample Incubation &amp; Lysis
2. Place tubes in tube rack on a rotating or gently shaking surface in a 56°C incubator
Secure rack with tape
Turn on rotator
Incubate for 90 minutes or overnight
After incubation proceed with steps below. Alternatively samples can be kept at 4°C overnight for a later extraction.
SECTION: Magnetic Beads Addition
3. After incubation, transfer 400 μl of the supernatant lysis to a new DWP (this is the sample plate). Bubble formation sometimes occurs due to the presence of SDS. Ensure the tubes are tightly sealed to prevent evaporation during the 56°C incubation period
Add 320 μl of Illumina Tune beads to the sample plate wells (1:8 sample to beads ratio to remove small DNA fragments < 200bp)
SECTION: Sample Incubation &amp; Lysis
2.1. Washing and Elution Plates Preparation
During the 90 minute incubation step, prepare two wash plates by pipetting 500 μl freshly prepared 80% ethanol into the wells of two DWPs
Prepare an elution plate by pipetting 100 μL of 10 mM Tris-HCl into the wells of a 200 μl plate
SECTION: KingFisher Extraction (DNA Purification)
4. Place the sample plate (containing the 720 μl of sample + beads), wash plates 1 and 2 and the elution plate into the KingFisher and run the program as follows
The KingFisher is optimized for the volumes stated in this protocol
SECTION: KingFisher Extraction (DNA Purification)
4.1. KingFisher Program

1. Pick up:
   Tip comb plate
2. Binding:
   Mix for 5 minutes at medium speed
   Collect beads, count: 5
   Move to sample plate
3. 1st Wash:
   Release beads
   Bottom mix and medium speed, each for 10 seconds, loop count 2
   Collect beads, count: 3; collect time: 5 seconds
   Transfer to wash plate 1
4. 2nd Wash:
   Release beads for 20 seconds
   Bottom mix and medium speed, each for 10 seconds, loop count 2
   Collect beads, count: 4; collect time: 5 seconds
   Transfer to wash plate 2
5. Dry:
   Use wash plate 2
6. Elution:
   Preheat to 60°C
   Bottom mix for 15 seconds, medium speed for 45 seconds, loop count: 6
   Collect beads, count: 1
   Transfer to elution plate
7. Collection of Beads:
   2 minutes at slow speed