Oct 13, 2022

Public workspaceDNA extraction for fermented plant based foods

DOI
dx.doi.org/10.17504/protocols.io.36wgqj9wkvk5/v1
  • Anique Ahmad1,
  • Arya Gautam1,
  • Tsedenia Denekew1,
  • Ahmed Shibl1,
  • Aashish Jha1
  • 1New York University, Abu Dhabi
  • Anique Ahmad: aa5506@nyu.edu
  • Aashish Jha: jhaar@nyu.edu
nyuad.geneticheritagegroup
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DOI: dx.doi.org/10.17504/protocols.io.36wgqj9wkvk5/v1
Protocol Citation: Anique Ahmad, Arya Gautam, Tsedenia Denekew, Ahmed Shibl, Aashish Jha 2022. DNA extraction for fermented plant based foods. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj9wkvk5/v1
Manuscript citation:
Anique Ahmad, Arya Gautam, Tsedenia Denekew, Ahmed, Shibl, Aashish Jha (2022). DNA extraction from fermented plant based foods. protocols.io
dx.doi.org/10.17504/protocols.io.36wgqj9wkvk5/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
Created: September 19, 2022
Last Modified: October 13, 2022
Protocol Integer ID: 70218
Funders Acknowledgement:
The Bill and Melinda Gates Foundation
Abstract
DNA Extraction for fermented plant based foods
Guidelines
Disinfect mortar and pestle with 70% ethanol in between samples
If the goal is metagenomic sequencing, we recommend bypassing the mortar and pestle step. Simply suspend the samples in 1 ml of ZymoBIOMICS™ Lysis Solution, shake vigorously or vortex, and transfer 750 uL of the supernatant to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and proceed to Step 2.
Materials
ZymoBIOMICS DNA Extraction Kit
Fecal samples
Mortar and pestle
Liquid nitrogen
Ice box with dry ice
1.5 ml microcentrifuge tubes
Vortex
Omni Bead Ruptor Elite bead beater
Centrifuge
Safety warnings
Handle liquid nitrogen with proper training and PPE
Before start
  • Label the following:
ZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm)
ZymoSpinTM III-F Filter in Collection Tube
Collection Tube
ZymoSpinTM II-CR Column in Collection Tube
ZymoSpinTM III-HRC Filter in Collection Tube
2 Sets of 1.5 ml microcentrifuge tubes (not provided with kit)
  • Spin the labelled ZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm) for 10 seconds in a mini-centrifuge to ensure that the beads have settled at the bottom
  • Include 1 control per batch and assign its position randomly.
Take Amount250 mg of food sample while on dry ice and transfer to mortar. Add liquid nitrogen until food sample is completely immersed. Using pestle vigorously homogenize sample until a powder forms. Transfer all of the sample to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm). AddAmount750 µL ZymoBIOMICS™ Lysis Solution to the tube. Cap tightly.

Secure in a Omni Bead Ruptor Elite bead beater fitted with a 2 ml tube holder assembly and process at max speed (30 m/s) for Duration00:05:00 . Rest for Duration00:05:00 .
10m
Centrifuge the ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) in a microcentrifuge at ≥ Centrifigation10000 x g, 00:01:00 .
1m
Transfer up to Amount600 µL supernatant to the Zymo-Spin™ III-F Filter in the labelled Collection Tube and centrifuge at Centrifigation8.000 x g, 00:01:00 . Discard the Zymo-Spin™ III-F Filter.
1m
Add Amount600 µL of ZymoBIOMICS™ DNA Binding Buffer to the filtrate in the labelled Collection Tube from Step 4. Mix well.
Repeat Step 5 so that the final volume of ZymoBIOMICS™ DNA Binding Buffer added is Amount1200 µL .
Transfer Amount800 µL of the mixture from Step 5 to a Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at Centrifigation10.000 x g, 00:01:00 .

1m
Discard the flow through from the Collection Tube and repeat Step 6.
Add Amount400 µL ZymoBIOMICS™ DNA Wash Buffer 1 to the Zymo-Spin™ IICR Column in a new Collection Tube and centrifuge at Centrifigation10.000 x g, 00:01:00 . Discard the flow-through.

1m
Add Amount700 µL ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at Centrifigation10.000 x g, 00:01:00 . Discard the flow-through.

1m
Repeat with Amount200 µL ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at Centrifigation10.000 x g, 00:01:00 .

1m
Transfer the Zymo-Spin™ IICR Column to a clean 1.5 ml microcentrifuge tube and add Amount100 µL (50 µl minimum) ZymoBIOMICS™ DNase/RNase Free Water directly to the column matrix and incubate for Duration00:05:00 . Centrifuge at Centrifigation10.000 x g, 00:01:00 to elute the DNA

6m
Place a Zymo-Spin™ III-HRC Filter in a new Collection Tube and add Amount600 µL ZymoBIOMICS™ HRC Prep Solution. Centrifuge at Centrifigation8.000 x g, 00:03:00 .

3m
Transfer the eluted DNA (Step 10) to a prepared Zymo-Spin™ III-HRC Filter in a clean 1.5 ml microcentrifuge tube and centrifuge at exactly Centrifigation16.000 x g, 00:03:00 .
3m
The filtered DNA is now suitable for PCR and other downstream applications. Eluted DNA should be frozen (–30 to –15°C or –90 to –65°C)