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

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.

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

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)

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

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

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.