HTTM : gDNA extraction

Prepare the lysis solution by adding 165µL of proteinase K to 66mL of homemade lysis buffer and mix well.

Add 600µLof lysis solution to each well of the deep-well plate and resuspend the pellet.

Cover with an adhesive aluminum foil and incubate at 55°C for 1h 0m 0s.

While still warm, add 260µLof ethanol 100%, without overmixing.

Transfer immediately to a deep-well plate fitted with an array of silica columns.

Centrifuge twice at 4000x g .

Discard flowthrough and add 500µL of wash solution.

Centrifuge at 3000x g

Repeat steps 7 and 8.

Discard flowthrough.

Centrifuge at 3000x g to eliminate traces of wash solution.

Discard flowthrough.

Add a collector plate between the silica column array and the deep-well plate.

Add 50µLof low TE to the silica matrix in each well.

Cover with an adhesive aluminum foil and incubate at55°C for 0h 15m 0s.

Centrifuge at 3000x g .

Put the contaminated silica array on an empty deep-well plate.

Add 150µL of 1N NaOH + 0.15%(v/v) Triton X-100 to each well.

Incubate at Room temperature for 0h 5m 0s

Centrifuge 3000x g

Add 200µL of 1,5N HCl+ 0,15% (v/v) Triton X-100 to each well.

Incubate at Room temperature for 0h 30m 0s

Centrifuge 3000x g

Add 150µL of 1N NaOH + 0,15%(v/v) Triton X-100 to each well.

Incubate at Room temperature for 0h 5m 0s

Centrifuge 3000x g

Collect the flowthrough in a beaker. Neutralize pH if needed and dispose of the flow through.

Add 200µLof ddH₂O to each well.

Centrifuge 3000x g

Repeat steps 25 and 26.

Silica columns array are ready to be reused.