DNA extraction from fecal samples

Phosphate-buffered saline (PBS)* EZ-beads (Promega, AMR76813M): 2 mL tube containing 0.2 mm ceramic (zirconium dioxide) spheres and one 5 mm zirconia bead

• Maxwell RSC Blood DNA Kit (Promega, AS1400)
• MN Bead Tube Holder (MACHEREY-NAGEL, 740469): Rubber-foam adapter for processing bead tubes with Vortex-Genie instrument

Maxwell RSC (Promega, AS4500): Automated nucleic acid purification platform* Micro Smash (TOMY, MS-100): Beads cell disrupter

• Vortex-Genie Mixer
• Heating block
• Microcentrifuge

Place 50-100 mg of fecal sample into tube.

Add 1 ml of PBS per 100 mg of feces.

Mix thoroughly by vortexing or pipetting.

Allow the sample to stand for 2 min to sediment large debris.

Transfer 300 µl of the suspension to 1.5 mL tube.

Centrifuge at 18,000 x g for 3 min.

Discard the supernatant.

Resuspend the pellet (~30 mg of feces) in 300 µl of PBS.

Incubate at 70°C for 10 min and cool to room temperature.

Transfer 300 µl of the suspension to EZ-beads tube.

Lyse cells either by using disruption device (13.1) or vortex mixer (13.2).

Place the EZ-beads tube in Micro Smash instrument and disrupt cells at 2,500 rpm for 2 min.

Place the EZ-beads tube on MN Bead Tube Holder attached to Vortex-Genie mixer and vortex for 5 min at maximum speed.

Briefly spin the tube.

Add 300 µl of Lysis Buffer and 30 µl of Proteinase K Solution to the sample in EZ-beads tube.

Mix thoroughly by vortexing for 10 sec.

Incubate at 56°C for 20 min.

Briefly spin the tube.

Transfer the supernatant (~600 µl) to 1.5 mL tube.

Centrifuge at 18,000 x g for 3 min.

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.