DNA extraction from rectal mucosa biopsies and matched faecal samples taken from surgery for microbiome analysis

Label and weigh a 2mL Matrix E tube tube (MPBio). Transfer stool (approximately 0.2g ) and weigh to record weight of stool sample to be extracted.

Add 928µL Sodium Phosphate buffer; 122µL MT buffer (both from MPBio Kit) to the sample in the Matrix E tube.

Perform Bead Beating with the MP Biomedicals FastPrep-24 for 0h 1m 0s at 5m/s.

Incubate at 85°C for 0h 15m 0s on the heating block.

Repeat steps 3 and 4.

Centrifuge at 14000rpm,0h 0m 0s for 0h 5m 0s and transfer supernatant to a fresh 2ml Eppendorf tube.

Add 250µL PPS Solution (MPBio), mix by inverting tube 12 times.

Centrifuge at 14000rpm,0h 0m 0s for 0h 5m 0s and transfer supernatant to a 15ml tube.

Vortex the Binding Matrix (MPBio) before use to ensure matrix is fully resuspended and add 1mL to the 15ml tube.

Mix by inverting the 15ml tubes for 0h 2m 0s then leave standing for 0h 5m 0s to collect the binding matrix at the bottom of the 15ml tube.

Remove and discard 500µL of supernatent without disturbing the Binding Matrix at the bottom of the tube.

Gently resuspend the Matrix in the remaining solution by pipetting up and down and transfer 600µL to a spin filter column (MPBio).

Centrifuge at 14000rpm,0h 0m 0s for 0h 1m 0s then empty the flow through from the tube.

Repeat steps 12 and 13 if necessary until all the Binding Matrix solution is applied to the spin filter column.

Add 500µL SEWSM wash buffer (MPBio) to the spin column, pipette gently until the Matrix is fully resuspended. (Ensure SEWSM buffer has 100% Ethanol added according to the kit instructions before use)

Centrifuge at 14000rpm,0h 0m 0s for 0h 1m 0s. Discard the flow through and centrifuge at 14000rpm,0h 0m 0s for 0h 2m 0s to dry.

Air dry for 0h 5m 0s then transfer the spin column to a fresh, labelled catch tube (MPBio).

Apply 80µL DES elution buffer to the Matrix in the spin column and pipette gently up and down to resuspend the Matrix. (Heating the DES buffer on the block for at before using may make this easier). 0h 5m 0s at 55°C before using may make this easier).

Centrifuge 14k for 0h 1m 0s to collect eluted sample. Remove the spin column and store the DNA sample tube for QC.

Label and weigh a 2ml Eppendorf tube. Transfer rectal mucosa biopsy tissue in to the 2ml Eppendorf tube. Re-weigh to calculate weight of biopsy tissue and record. (Aim for 5-60mg of starting tissue).

Add 828µL Sodium Phosphate buffer; 122µL MT buffer (both from MPBio Kit), 50µL Lysozyme (100mg/ml) and 50µL Mutanolysin (5U/μl) to the tube, briefly vortex to mix and incubate on a heat block at 37°C for 1h 0m 0s

Transfer entire contents of the Eppendorf tube to a Matrix E tube (MPBio Kit).

Perform Bead Beating with the MP Biomedicals FastPrep-24 for 0h 1m 0s at 5m/s.

Incubate at 85°C for 0h 15m 0s on the heating block.

Repeat steps 23 and 24 .

Centrifuge at 14000rpm,0h 0m 0s for 0h 5m 0s and transfer supernatant to a fresh 2ml Eppendorf tube.

Add 250µL PPS Solution (MPBio), mix by inverting tube 12 times.

Centrifuge at 14000rpm,0h 0m 0s for 0h 5m 0s and transfer supernatant to a 15ml tube.

Vortex the Binding Matrix (MPBio) before use to ensure matrix is fully resuspended and add 1mL to the 15ml tube.

Mix by inverting the 15ml tubes for 0h 2m 0s then leave standing for 0h 5m 0s to collect the binding matrix at the bottom of the 15ml tube.

Remove and discard 500µL of supernatent without disturbing the Binding Matrix at the bottom of the tube.

Gently resuspend the Matrix in the remaining solution by pipetting up and down and transfer 600µL to a spin filter column (MPBio).

Centrifuge at 14000rpm,0h 0m 0s for 0h 1m 0s then empty the flow through from the tube.

Repeat steps 32 and 33 if necessary until all the Binding Matrix solution is applied to the spin filter column.

Add 500µL SEWSM wash buffer (MPBio) to the spin column, pipette gently until the Matrix is fully resuspended. (Ensure SEWSM buffer has 100% Ethanol added according to the kit instructions before use)

Centrifuge at 14000rpm,0h 0m 0s for 0h 1m 0s. Discard the flow through and centrifuge at 14000rpm,0h 0m 0s for 0h 2m 0s to dry.

Air dry for 0h 5m 0s then transfer the spin column to a fresh, labelled catch tube (MPBio).

Apply 80µL DES elution buffer to the Matrix in the spin column and pipette gently up and down to resuspend the Matrix. (Heating the DES buffer on the block for at before using may make this easier). 0h 5m 0s at 55°C before using may make this easier).

Centrifuge 14k for 0h 1m 0s to collect eluted sample. Remove the spin column and store the DNA sample tube for QC and microbial DNA enrichment.