DNA extraction from insect gut-dwelling fungi

Suspend fungal thalli and spores in a 1.5 mL centrifuge tube with 2x CTAB buffer (200µL).

Freeze and thaw the sample three times by submerging the tube into liquid nitrogen and incubating it at 65°Cusing a heat block.

After the final thaw, crush the fungal tissues using a disposable pellet pestle (for 1.5 mL microtube).

Cool the sample for 0h 1m 0s On ice.

Add 4µL Proteinase K 20mg/mL to the tube and mix them by inverting the tube three times.

Warm the tube at 65°Cfor 0h 30m 0s using a heat block.

Centrifuge at 12000x g and transfer the supernatant to a new tube.

Add Chloroform: Isoamyl alcohol (24:1) with an equal volume of the supernatant.

Shake the mixture slowly for 0h 10m 0s on a rotator and leave the tube on the rack for 0h 2m 0s at room temperature.

Centrifuge at 13000x g and transfer the top layer to another tube with wide-bore tips, avoiding breaking DNAs.

Transfer the supernatant to a new tube and repeat steps 8-10.

Add 4µL RNase A (10mg/mL) and incubate at 37°C for 0h 40m 0s.

Add Isopropanol ( stored at ) -20°C) with a 66% volume of the product from the last step and mix by inversion.

Leave the tube at 4°C for at least 5h 0m 0s (or overnight) until fully precipitated.

Centrifuge at 13000x g with the cap opening side pointing to the centrifuge center.

Immediately dump the supernatant and transfer the tube (with the cap open & upside down) on the paper towel to absorb the remaining supernatant.

Leave the cap open on rack.

Add 150µL 70% ethanol (stored at -20°C) to the tube.

Cap it and use the finger to tap the tube gently.

Centrifuge at 13000x g.

Repeat step 16.

Leave the tube at 37°C with the cap open and a piece of Kimwipe paper on top for 0h 30m 0s or until the DNA becomes dry.

Add 50µL H₂O to the dried DNA in the tube and wait for 0h 10m 0s.

Pipette and store the DNA in the tube at -20°C.