Nov 09, 2022

Public workspaceDNA extraction from fecal samples

DOI
dx.doi.org/10.17504/protocols.io.5jyl8jnorg2w/v1
  • Anique Ahmad1,
  • Arya Gautam1,
  • Tsedenia Denekew1,
  • Aashish Jha1
  • 1New York University, Abu Dhabi
  • Aashish Jha: jhaar@nyu.edu
nyuad.geneticheritagegroup
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DOI: dx.doi.org/10.17504/protocols.io.5jyl8jnorg2w/v1
Protocol Citation: Anique Ahmad, Arya Gautam, Tsedenia Denekew, Aashish Jha 2022. DNA extraction from fecal samples. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jnorg2w/v1
Manuscript citation:
Anique Ahmad, Arya Gautam, Tsedenia Denekew, Aashish Jha (2022). DNA extraction from fecal samples. protocols.io
dx.doi.org/10.17504/protocols.io.5jyl8jnorg2w/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: September 16, 2022
Last Modified: November 09, 2022
Protocol Integer ID: 70128
Abstract
DNA Extraction from fecal samples
Guidelines
Table 1. Sample type and maximum input for DNA extraction using this protocol

Materials
ZymoBIOMICS DNA Extraction Kit
Fecal samples
Homogenizing pestle
Liquid nitrogen or dry ice
Ice box with ice
1.5 ml microcentrifuge tubes
Vortex
Omni Bead Ruptor Elite bead beater
Centrifuge
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.
Transfer fecal sample to ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and add ZymoBIOMICS™ Lysis Solution:

  • Flash frozen fecal samples will be solid and an aliquot (Amount15 mg ) of feces is expected in a 2mL container (Table 1). Add Amount250 µL of ZymoBIOMICS™ Lysis Solution to the fecal sample and homogenize evenly using the homogenizing pestle while submerged in liquid nitrogen (or on dry ice). Once the sample is homogenized, add another Amount500 µL of ZymoBIOMICS™ Lysis Solution such that the final volume is Amount750 µL . Mix well by vortexing.
  • Spin the sample container for Duration00:00:10 in a minicentrifuge.
  • Transfer the entire Amount750 µL of the sample mix into the ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) and proceed to Step 2.
10s
Fecal samples collected using Zymo DNA/RNA Shield™ will exist as a solution (Table 1). Mix the sample by shaking well or vortexing. Add up to Amount250 µL of such sample to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm). Adjust final volume to Amount1 mL with ZymoBIOMICS™ Lysis Solution. Cap the tube tightly and proceed to Step 2.
Fecal samples collected using other stabilization kits may exist as a solution (Table 1). Mix the sample by shaking well or vortexing. Add Amount250 µL of such sample to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm). AddAmount750 µL ZymoBIOMICS™ Lysis Solution to the tube. Cap tightly and proceed to Step 2.
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 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)