ZymoBIOMICS‱ DNA Extraction (following the manufacturer’s instruction except for beat beating time) (reference no. 1)
1.1. Add 750 µl ZymoBIOMICS‱ Lysis Solution to the ZR BashingBead‱ Lysis Tubes.
1.2 Pick up bacterial isolates (whole plate) using inoculate loop and add them to the tube and cap tightly.
1.3. Secure in a bead beater or vortex with a 2 ml tube and process at maximum speed for 3 minutes (modified from original protocol which suggests to beat for 20 minutes).
1.4. Centrifuge the ZR BashingBead‱ Lysis Tubes in a microcentrifuge at ≥ 10,000 x g for 1 minute.
1.5. 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. Discard the Zymo-Spin‱ III-F Filter.
1.6. Add 1,200 μl of ZymoBIOMICS‱ DNA Binding Buffer to the filtrate in the Collection Tube (from Step 1.5 Step 1.5). Mix well.
1.7. 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.
1.8. Discard the flow through from the Collection Tube and repeat Step 1.7 repeat Step 1.7.
1.9. 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.
1.10. 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.
1.11. 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.
1.12. 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.
1.13. 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.
1.14. Transfer the eluted DNA (Step 1.12 Step 1.12) 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.