DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)

Add beta-mercaptoethanol (not included in kit) to the Genomic Lysis Buffer (Cat #: D3004-1-100) to a final dilution of 0.5% (v/v) (v/v) i.e., 2.5 ml per 500 ml for optimal performance.

Add 5g of soil sample to the bead/filter of a ZR Bashing Bead Lysis/Filtration Tube (50 mL w/ 0.5 mm Beads; Cat #: S6010) .

Add 6mL of BashingBead Buffer (Cat #: D6001-3-150) to the sample. Cap tube tightly and secure with parafilm.

Place tubes in a bead beater (Bullet Blender) and process the samples for 10 minutes at speed 8.

Tip: Store tubes sideways until they go into the bead beater to prevent liquid from prematurely going through filter.

Tip: Select "Zymo MidiPrep" pre-loaded protocol on Bullet Blender in Bik Lab.

Centrifuge the ZR BashingBead Lysis/Filtration Tube in a centrifuge at 5000rpm.

Remove bead/filter chamber from the top of the ZR BashingBead Lysis/Filtration Tube and transfer supernatant from the bottom of the tube (using a 10 mL pipette) to a new 50 mL tube.

Add 18mL of Genomic Lysis Buffer to the supernatant. Mix well.

Tip: Centrifuge for a few seconds if there is too much foam.

Filter the entire mixture using a Zymo-Spin V-E Column/Zymo-Midi Filter (Cat #: C1021-25) mounted on a vacuum manifold, at ≥600 mm Hg.

Disconnect the Zymo-Spin V-E Column/Zymo-Midi Filter and transfer the Zymo-Spin V-E Column to a Collection Tube (Cat #: C1001-50) . Spin the column at 10000rpmin a microcentrifuge.

Add 300µL of DNA Pre-Wash Buffer (Cat #: D3004-5-15) to the column and spin at 10000rpm. Discard the flow through.

Tip: Add reagent by inserting the pipette tip at an angle while pressing it against the wall of the column and without touching the filter to avoid overflow.

Add 400µL of g-DNA Wash Buffer (Cat #: D3004-2-50) to the column and centrifuge at 10000rpm. Discard the flow through.

REPEAT WASH STEP: Add 400µL of g-DNA Wash Buffer to the column and centrifuge at 10000rpm. Discard the flow through.

Transfer the Zymo-Spin V-E Column to a 1.5 mL microcentrifuge tube. Add 150µL of DNA Elution Buffer (Cat #: D3004-4-16) directly to the column matrix. Wait for 1 minute and then centrifuge at 10000rpm to elute the DNA.

Place the Zymo-Spin III-HCR Filter in a clean Collection Tube . Add 600µL of Prep Solution (Cat #: D6035-1-30) . Centrifuge at 8000rpm.

Place the Zymo-Spin III-HCR Filter in a clean 1.5 mL microcentrifuge tube. Transfer the eluted DNA to the prepared Zymo-Spin III-HCR Filter and centrifuge at exactly 16000rpm. The filtered DNA is now suitable for PCR and other downstream applications.