ZymoBIOMICS™ DNA Extraction (following the manufacturer’s instruction except for beat beating time) (reference no. 1)
1.1. Transfer 5-10 ml collected saliva/ mouthwash sample to 15 ml sterile conical centrifuge tube
1.2. Centrifuge the tube at 4000 rpm for 20 minutes at 4°C
1.3. Discard supernatant as much supernatant as possible. Save the pellet
1.4. Add 750 µl ZymoBIOMICS™ Lysis Solution to the tube that contain pellet and mix the lysis buffer with the pellet.
1.5. Move all buffer to ZR BashingBead™ Lysis Tubes and cap tightly.
1.6. Secure in a bead beater or vortex with a 2 ml tube and process at maximum speed for 2-3 minutes (modified from original protocol which suggests to beat for 20 minutes).
1.7. Centrifuge the ZR BashingBead™ Lysis Tubes in a microcentrifuge at ≥ 10,000 x g for 1 minute.
1.8. Transfer up to 400 µl supernatant to the Zymo-Spin™ III-F Filter in a Collection Tube and centrifuge at 8,000 x g for 1 minute. Discard the Zymo-Spin™ III-F Filter.
1.9. Add 1,200 μl of ZymoBIOMICS™ DNA Binding Buffer to the filtrate in the Collection Tube (from Step 1.8). Mix well.
1.10. Transfer 800 µl of the mixture from Step 5 to a Zymo-Spin™ IIC-Z Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute.
1.11. Discard the flow through from the Collection Tube and repeat Step 1.10.
1.12. Add 400 µl ZymoBIOMICS™ DNA Wash Buffer 1 to the Zymo-Spin™ IIC-Z Column in a new collection Tube and centrifuge at 10,000 x g for 1 minute. Discard the flow-through.
1.13. Add 700 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IIC-Z Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute. Discard the flow-through.
1.14. Add 200 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IIC-Z Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute.
1.15. Transfer the Zymo-Spin™ IIC-Z Column to a clean 1.5 ml microcentrifuge tube and add 100 µl (50 µl minimum) NFW directly to the column matrix and incubate for 1 minute. Centrifuge at 10,000 x g for 1 minute to elute the DNA.
1.16. 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.
1.17. Transfer the eluted DNA (Step 1.15) to a prepared Zymo-Spin™ III-HRC Filter in a clean 1.5 ml microcentrifuge tube and centrifuge at 16,000 x g for 3 minutes. The filtered DNA is now ready for the cleanup step.