DNA extraction protocol for DNA-metabarcoding of marine phytoplankton using Zymobiomics DNA minprep kit (Zymo Research; D4300)

Filter water samples onto 0.2 µm pore size Millipore filter (Merck Millipore; GSWP04700) attached to a filter funnel connected to a vacuum source. Filter a minimum of 500 ml collected seawater.

500mL

Using two sets of sterile forceps, pick up the filter membrane at opposite edges and roll the filter into a 2 ml cryo tube (Avantor, 479-1262) with the top side of the filter facing inward.

Take note of station, date, sample ID, sampling time, time duration of filtration, filtered volume and any comments of potential use (such as filtration problems, colour of filter etc.).

Store the filters at minimum -20°C (short-term storage) or -80°C (long-term storage) no additional reagent for preservation is needed.

-20°C

Clean your work bench with 70% ethanol.* Prepare sterilized forceps and petri dish on the bench.

• Prepare pipettes in varying sizes with filter tips.
• Take out beakers for discarding liquids and used tips.
• Turn on the microcentrifuge and set the machine to 13000 x g for 1 minute.
• Prepare Vortex with adapter plates and set timer for 10 minutes.

Prepare spike-in genes using artificial 16S and 18S rRNA gene oligonucelotides (designed in-house and generated by Twist Bioscience). See sequences and sub-steps below:

Add 100 µl sterile filtered autoclaved MQ, DNase/RNase Free Water or TE buffer to the dry pellet bringing the stock solution to a concentration of 10 ng/µl.

100µL

Then, in sequence through continued serial dilution in steps of x 10 dilutions, prepare working solutions with the final concentration of the 16S and 18S spike-in DNA to 0.0002 ng/µl and 0.0007 ng/µl , respectively, note the x 10 dilution for the final concentration compared to Table 2.

Aliquot and freeze working solution in batches at -20°C until processing.

-20°C

Thaw filters in the sampling tubes at room temperature and add the filter cut into small pieces to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) .

Room temperature

Rinse the sampling tube with 750 µl and add the Lysis Solution into the ZR BashingBead™ Lysis Tube .

750µL

Note: For samples stored and lysed in DNA/RNA Shield™ Lysis buffer , do not add ZymoBIOMICS™ Lysis Solution and proceed to step 9.

Add 10 µl of the prepared dilutions of 16S and 18S spikes , and cap tightly.

10µL

Secure in a Vortex-Genie 2 fitted with a 2 ml tube holder assembly (adapter) and process at maximum speed for 10 minutes.

3200rpm

Note: Vortex for 10 min at maximum speed (2850Hz) on a Vortex-Genie 2 is a specific selected step and slight modification from the standard ZymoBIOMICS protocol.

Centrifuge the ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) in a microcentrifuge at 13,000 x g for 1 minute.

13000x g

Transfer up to 400 µl supernatant to the Zymo-Spin™ III-F Filter (red mark) in a Collection Tube and centrifuge at 8,000 x g for 1 minute. Discard the Zymo-Spin™ III-F Filter .

400µL

8000x g

Binding preparation: Add 1,200 µl of ZymoBIOMICS™ DNA Binding Buffer to the filtrate in the Collection Tube from Step 12. Mix well.

1200µL

Transfer 800 µl of the mixture from Step 13 to a Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute.

10000x g

Discard the flow through from the Collection Tube and REPEAT STEP 14 .

Place the Zymo-Spin™ IICR Column in a NEW Collection Tube NEW Collection Tube.

Add 400 µl ZymoBIOMICS™ DNA Wash Buffer 1 to the Zymo-Spin™ IICR Column and centrifuge at 10,000 x g for 1 minute. Discard the flow-through.

400µL

10000x g

Add 700 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute. Discard the flow-through.

700µL

10000x g

Add 200 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute.

200µL

10000x g

Transfer the Zymo-Spin™ IICR Column to a clean 1.5 ml microcentrifuge tube and add 50 µl ZymoBIOMICS™ DNase/RNase Free Water directly to the center of the column matrix and incubate in room temperature for 1 minute. Centrifuge at 10,000 x g for 1 minute to elute the DNA.

50µL

Room temperature

10000x g

Note: Adding 50 μl of ZymoBIOMICS DNase/RNase Free Water is a specific selected step and slight modification from the standard ZymoBIOMICS protocol.

Place a Zymo-Spin™ III-HRC Filter in a NEW Collection Tube and add 600 µl ZymoBIOMICS™ HRC Prep Solution . Centrifuge at 8,000 x g for 3 minutes.

600µL

8000x g

Place the prepared Zymo-Spin™ III-HRC Filter in a clean 1.5 ml microcentrifuge tube 1.5 ml microcentrifuge tube. Make sure to mark the tube (both lid and side) clearly with a unique ID using a permanent marker resistant to freezing.

Transfer the eluted DNA (Step 20) to the Zymo-Spin™ III-HRC Filter and centrifuge at exactly 16,000 x g for 3 minutes.

16000x g

Freeze extracted gDNA at -20°C or -80°C. Measure the concentration of the extracted DNA using Qubit Fluorometer ; see separate protocol for Qubit provided by the manufacturer. The filtered DNA is now suitable for PCR and other downstream applications.

-20°C