TITLE: The &#39;Three Peaks&#39; faecal DNA extraction method for long-read sequencing
AUTHORS: Josh Quick

[STEPS]
?. Add  fresh stool or  OMNIgene GUT to DNA/RNA Shield, vortex briefly and place on a tube rotator for  at .
100 mg
50 µl
200 µl
Centrifuge: 20 33
?. Centrifuge at  for  and retain up to  supernatant depending on size of pellet.
Centrifuge: 5000 34
200 µl
?. Add  PBS and resuspend material by pipetting up and down, centrifuge at  and retain up to  supernatant depending on size of pellet.
100 µl
Centrifuge: 5000 34
100 µl
?. Add  PBS and resuspend material by pipetting up and down, centrifuge at  for  and discard supernatant.
1 ml
Centrifuge: 5000 34
?. Add  PBS and  MetaPolyzyme, mix by pipetting and incubate at  for . Gently mix by pipetting up and down to ensure solution is homogeneous before proceeding.
100 µl
5 µl
35 °C
?. Add  DNA/RNA Shield,  SDS and  Proteinase K and mix by pipetting. Incubate at  for  with  mixing. Gently mix by pipetting up and down to ensure solution is homogeneous before proceeding.
100 µl
10 µl
10 µl
55 °C
Centrifuge: 300 33
?. Centrifuge at  for  and retain up to  supernatant depending on size of pellet.
Centrifuge: 5000 34
200 µl
?. Resuspend pellet in  Lysis Solution and transfer to a ZR BashingBead Lysis Tube. Bead-beat on a FastPrep instrument for 1 cycle of  at
750 µl
6
?. Centrifuge tube at  for  and retain  supernatant.
Centrifuge: 10000 34
400 µl
?. Pool the supernatants retained at each of the steps in a 2 ml Eppendorf tube and measure the volume using a P1000 pipette.
?. Add 2 volumes of Genomic Lysis Buffer and  MagBinding beads and place on a tube rotator for  at .
50 µl
Centrifuge: 20 33
?. Put tube on a magnetic rack and incubate for  or until clear. Discard supernatant taking care not to disturb bead pellet.
?. Remove from the magnetic rack and using a P1000 pipette add  DNA elution buffer. Mix by pipetting up and down x10.
100 µl
?. Add  Quick-DNA MagBinding Buffer and place on a tube rotator for  at .
500 µl
Centrifuge: 20 33
?. Put tube on a magnetic rack and incubate for  or until clear. Discard supernatant taking care not to disturb bead pellet.
?. Remove from the magnetic rack and using a P1000 pipette add  DNA Pre-Wash Buffer. Mix by pipetting up and down x10 and transfer to a new Eppendorf tube.
900 µl
1.5 ml
?. Put tube on a magnetic rack and incubate for  or until clear. Discard supernatant taking care not to disturb bead pellet.
?. Remove from the magnetic rack and using a P1000 pipette add  g-DNA Wash Buffer. Mix by pipetting up and down x10 and transfer to a new Eppendorf tube.
900 µl
1.5 ml
?. Put tube on a magnetic rack and incubate for  or until clear. Discard supernatant taking care not to disturb bead pellet.
?. and repeat wash with g-DNA Wash Buffer for a second time.
?. Using a P1000 wide-bore pipette tip to reduce turbulence add  DNA elution buffer to the front of the tube and immediately remove it again. Try to do this in a fast, smooth motion disturbing the beads as little as possible.
900 µl
?. Remove the residual buffer from the bottom of the tube and discard.
?. With a P1000 pipette add  DNA elution buffer to the tube and mix by pipetting up and down x10.
50 µl
?. Place on a tube rotator for  at .
Centrifuge: 20 33
?. Put tube on a magnetic rack and incubate for  or until clear. Transfer DNA to a new Eppendorf tube. Store DNA at  for up to a month or use immediately.
1.5 ml
5 °C