TITLE: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
AUTHORS: Jenna Brown, Mirayana Marcelino Barros, Tiago Pereira, Alejandro De Santiago Perez, Hunter Powell

[DESCRIPTION]
DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110); kit and protocol used for DNA extraction of soil samples in the Bik Lab. 

[STEPS]
SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
2. Add 5 g of soil sample to the bead/filter of a ZR Bashing Bead Lysis/Filtration Tube (50 mL w/ 0.5 mm Beads; Cat #: S6010).

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
3. Add 6 mL of BashingBead Buffer (Cat #: D6001-3-150) to the sample. Cap tube tightly and secure with parafilm.  

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
3.1. 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.
SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
3.2. Centrifuge the ZR BashingBead Lysis/Filtration Tube in a centrifuge at 5000 rpm, 5 min.

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
3.3. 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.
SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
4. Add 18 mL of Genomic Lysis Buffer to the supernatant. Mix well. 
Tip: Centrifuge for a few seconds if there is too much foam. 

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
4.1. 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.

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
4.2. 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 10000 rpm, 1 minin a microcentrifuge. 

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
5. Add 300 µL of DNA Pre-Wash Buffer (Cat #: D3004-5-15) to the column and spin at 10000 rpm, 1 min. 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.

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
6. Add 400 µL of g-DNA Wash Buffer (Cat #: D3004-2-50) to the column and centrifuge at 10000 rpm, 1 min. Discard the flow through. 

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
8. Transfer the Zymo-Spin V-E Column to a 1.5 mL microcentrifuge tube. Add 150 µL of DNA Elution Buffer (Cat #: D3004-4-16) directly to the column matrix. Wait for 1 minute and then centrifuge at 10000 rpm, 1 min to elute the DNA.

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
9. Place the Zymo-Spin III-HCR Filter in a clean Collection Tube. Add 600 µL of Prep Solution (Cat #: D6035-1-30). Centrifuge at 8000 rpm, 3 min.

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
9.1. 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 16000 rpm, 3 min. The filtered DNA is now suitable for PCR and other downstream applications.

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
1. 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.

SECTION: DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
7. REPEAT WASH STEP: Add 400 µL of g-DNA Wash Buffer to the column and centrifuge at 10000 rpm, 1 min. Discard the flow through.