Aug 24, 2022

Public workspaceDNA extraction from fecal samples V.3

DOI
dx.doi.org/10.17504/protocols.io.3byl4k912vo5/v3
  • 1Kansai Medical University
Yoshiyuki Matsuo
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DOI: 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 21, 2022
Last Modified: August 24, 2022
Protocol Integer ID: 62955
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
Preparation of fecal samples
Place Amount50-100 mg of fecal sample into tube.
Add Amount1 mL of PBS per Amount100 mg of feces.

Pipetting
Mix thoroughly by vortexing.
Mix
Allow the sample to stand for Duration00:02:00 to sediment large debris.

2m
Incubation
Transfer Amount300 µL of the suspension to 1.5 mL tube.

Pipetting
Centrifuge at Centrifigation10000 x g, 00:03:00 .

3m
Centrifigation
Discard the supernatant.
Pipetting
Resuspend the pellet (~Amount30 mg of feces) in Amount300 µL of PBS.

Pipetting
Mix
Incubate at Temperature70 °C for Duration00:10:00 on the block heater.

10m
Incubation
Cool to TemperatureRoom temperature .

Mechanical cell disruption by bead beating
Mechanical cell disruption by bead beating
Transfer Amount300 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.

Pipetting
Lyse cells either by using disruption device (12.1) or vortex mixer (12.2).
Digestion
Place the EZ-beads tube in Micro Smash instrument and disrupt cells by shaking at Shaker2500 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 Duration00:05:00 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.
Centrifigation
Automated DNA extraction using Maxwell RSC Blood DNA Kit
Automated DNA extraction using Maxwell RSC Blood DNA Kit
23m
23m
Add Amount300 µL of Lysis Buffer and Amount30 µL of Proteinase K Solution to the sample in EZ-beads tube.

Pipetting
Mix by inverting the tube.
Mix
Briefly spin the tube.
Centrifigation
Incubate at Temperature56 °C for Duration00:20:00 on the block heater.

20m
Incubation
Digestion
Briefly spin the tube.
Centrifigation
Transfer the supernatant (~Amount500 µL ) to 1.5 mL tube.

Pipetting
Centrifuge at Centrifigation18000 x g, 00:03:00 .

3m
Centrifigation
Transfer the cleared lysate to Maxwell RSC Cartridge.
Pipetting
Add Amount50 µL of Elution Buffer to elution tube.

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