Aug 24, 2022

DNA extraction from fecal samples V.3

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4k912vo5/v3
  • 1Kansai Medical University
Yoshiyuki Matsuo
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl4k912vo5/v3
Protocol Citation: Yoshiyuki Matsuo 2022. DNA extraction from fecal samples. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4k912vo5/v3Version created by Yoshiyuki Matsuo
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 20, 2022
Last Modified: August 24, 2022
Protocol  Integer ID: 62955
Keywords: fecal samples dna extraction method, metagenomic sequencing, dna extraction, extraction, dna
Abstract
DNA extraction method for metagenomic sequencing of the gut microbiota
Materials
Reagents/Kits
  • Phosphate-buffered saline (PBS)

  • EZ-Beads (Promega/AMR, AMR76813M)

  • Maxwell RSC Blood DNA Kit (Promega, AS1400)


Equipment
  • Vortex mixer

  • High-speed microcentrifuge

  • Block heater

  • Micro Smash Beads Cell Disrupter (TOMY Digital Biology, MS-100)

  • Maxwell RSC instrument (Promega, AS4500)
Preparation of fecal samples
Place 50-100 mg of fecal sample into tube.
Add 1 mL of PBS per 100 mg of feces.

Mix thoroughly by vortexing.
Allow the sample to stand for 00:02:00 to sediment large debris.

2m
Transfer 300 µL of the suspension to 1.5 mL tube.

Centrifuge at 10000 x g, 00:03:00 .

3m
Discard the supernatant.
Resuspend the pellet (~30 mg of feces) in 300 µL of PBS.

Incubate at 70 °C for 00:10:00 on the block heater.

10m
Cool to Room temperature .

Mechanical cell disruption by bead beating
Transfer 300 µL of the suspension to EZ-beads tube.

Note
The EZ-Beads tube contains zirconium oxide beads of two different sizes (0.2 mm spheres and a large 5 mm bead) that can facilitate efficient cell lysis by bead beating.

Lyse cells either by using disruption device (12.1) or vortex mixer (12.2).
Place the EZ-beads tube in Micro Smash instrument and disrupt cells by shaking at 2500 rpm, 00:02:00 .
Equipment
Micro Smash Beads Cell Disrupter
NAME
TOMY Digital Biology
BRAND
MS-100
SKU

Safety information
Caution: Avoid using a disruption device with a high-speed linear reciprocating motion, as this may potentially result in breakage of the EZ-Beads tubes.


Place the EZ-beads tube on MN Bead Tube Holder attached to Vortex-Genie mixer and vortex for 00:05:00 at maximum speed.
Equipment
MN Bead Tube Holder
NAME
Rubber-foam adapter for processing bead tubes with Vortex-Genie instrument
TYPE
MACHEREY-NAGEL
BRAND
740469
SKU

5m
Briefly spin the tube to collect contents.
Automated DNA extraction using Maxwell RSC Blood DNA Kit
23m
Add 300 µL of Lysis Buffer and 30 µL of Proteinase K Solution to the sample in EZ-beads tube.

Mix by inverting the tube.
Briefly spin the tube.
Incubate at 56 °C for 00:20:00 on the block heater.

20m
Briefly spin the tube.
Transfer the supernatant (~500 µL ) to 1.5 mL tube.

Centrifuge at 18000 x g, 00:03:00 .

3m
Transfer the cleared lysate to Maxwell RSC Cartridge.
Add 50 µL of Elution Buffer to elution tube.

Start the extraction run following the manufacturer's instructions.
Equipment
Maxwell RSC instrument
NAME
Automated nucleic acid purification platform
TYPE
Promega
BRAND
AS4500
SKU