May 28, 2024
DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
- Jenna Brown1,
- Mirayana Marcelino Barros2,
- Tiago Pereira3,
- Alejandro De Santiago Perez2,
- Hunter Powell4
- 1University of Georgia- Holly Bik;
- 2University of Georgia;
- 3Department of Marine Sciences, University of Georgia;
- 4UGA
Protocol Citation: Jenna Brown, Mirayana Marcelino Barros, Tiago Pereira, Alejandro De Santiago Perez, Hunter Powell 2024. DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110). protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49b2ogo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 02, 2024
Last Modified: May 28, 2024
Protocol Integer ID: 99117
Funders Acknowledgement:
Holly Bik
Abstract
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.
DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
DNA Extraction: Zymo Research Quick-DNA Fecal/Soil Microbe Midiprep Kit (Cat #: D6110)
15m
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 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).
Add 6 mL 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 5000 rpm, 00:05:00 .
5m
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 18 mL 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 10000 rpm, 00:01:00 in a microcentrifuge.
1m
Add 300 µL of DNA Pre-Wash Buffer (Cat #: D3004-5-15) to the column and spin at 10000 rpm, 00:01:00 . 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.
1m
Add 400 µL of g-DNA Wash Buffer (Cat #: D3004-2-50) to the column and centrifuge at 10000 rpm, 00:01:00 . Discard the flow through.
1m
REPEAT WASH STEP: Add 400 µL of g-DNA Wash Buffer to the column and centrifuge at 10000 rpm, 00:01:00 . Discard the flow through.
1m
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, 00:01:00 to elute the DNA.
1m
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, 00:03:00 .
3m
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, 00:03:00 . The filtered DNA is now suitable for PCR and other downstream applications.
3m