DNA extraction from dermatophytes using the Macherey-Nagel NucleoSpin™ Blood QuickPure kit (REF: 740569)

Dissolve 30g of in

1L of and let mix on the heated magnetic stirrer for 0h 5m 0s (temperature and mixing speed knob at mid-step).

Cover the flask with glass wool and aluminium foil. Autoclave it at 121°C for 0h 30m 0s.

After allowing to cool, transfer 25mL of this medium into a tube. Label the tube with the strain number.

Using a sterile swab (or a sterile inoculation loop), gently collect the primary culture and dip the swab (or the sterile inoculation loop) into the tube containing the culture medium (prepared in the previous step). Close the tube halfway to allow gas exchange.

Allow to grow in the incubator at 30°C until a sufficient flocculate is formed (requires at least 96 hours). Incubation time varies from strain to strain but flocculate should be visible after 5 days. If this is not the case, repeat the cultivation step.

Preheat the elution buffer to 70°C

Using a Pasteur pipette, carefully remove the flocculate from the tube containing the previously cultured dermatophyte strain. Transfer this flocculate to a sterile tube containing glass beads, let's call it primary tube. Add 500µL of to the primary tube.

Cool this tube to -20°C on the ice block for 0h 1m 0s. Then, heat this tube in a water bath at 70°C for 0h 1m 0s. Finally, run this tube through the cell disruptor at maximum speed for 0h 1m 0s. This constitutes 1 cycle of 3 steps. You must repeat this cycle 5 times. The recovered mixture is referred to as primary lysate in the following steps.

Take 200µL of the primary lysate (from the preliminary steps) and transfer to a clean tube. Add 25µL of proteinase K and 200µL of lysis buffer BQ1. Homogenize with a vortex for 0h 0m 15s and then incubate the mixture for 0h 15m 0s at 70°C (in the water bath).

Add 200µL of absolute ethanol (96-100%), vortex for 0h 0m 15s and then short-spin centrifuge for MAXIMUM 0h 0m 10s at 11000x g to accelerate protein precipitation. Do not centrifuge any longer. This may cause the DNA to be lost from the supernatant in the pellet.

Gently collect the supernatant of the solution (approximately 600µL) then apply it in the chromatographic column with the silica membrane.

Centrifuge the column at 11000x g for 0h 1m 0s to allow absorption of DNA onto the silica membrane and removal of contaminants at the same time. Keep the column and discard the flows-through.

Place the column in a new collection tube and add 350µL of Buffer BQ2.Then centrifuge at 11000x g for 0h 3m 0s

Place the column in a new collection tube (or discard the flows-through) and add 200µL of Buffer BQ2. Then centrifuge again at 11000x g for 0h 1m 0s

Discard the flows-through or change the collection tube and centrifuge the column at 11000x g for 0h 1m 0s without adding any buffer.

Place the column in a clean collection tube. Add 50µL of elution buffer pre-heated at 70°C to the dried column.

Incubate for 0h 5m 0s at room temperature and then centrifuge for 0h 1m 0s at 11000x g Discard the column and keep the flows-though which is the purified DNA. Store DNA at -80°C to ensure stability.

To determine the purity and concentration of the DNA, a NanoDrop dosage was performed. For this purpose, a negative control was prepared beforehand. This control will have undergone all the extraction steps but will not contain any material from dermatophytes.

Launch the computer program and select the "nucleic acid" mode. Make sure the sample deposit spot is clean and dry. If necessary, clean it with the wipes provided for this purpose. Then drop 2µL of the negative control and click on the "blank" box.

Proceed in the same way to measure the sample containing the DNA, but click on "measure" instead of "blank". There is no need to redo a blank between measurements.