TITLE: DNA extraction from dermatophytes using the Macherey-Nagel NucleoSpin™ Blood QuickPure kit (REF: 740569)
AUTHORS: Khalid  El Moussaoui

[DESCRIPTION]
This protocol describes the steps necessary to extract and purify genomic DNA from dermatophytes (and more specifically from dermatophytes of the genus Trichophyton).

[GUIDELINES]
Perform the DNA dosage directly after extraction and not after a freeze/thaw cycle.
Be careful when preparing the medium : work under sterile conditions as much as possible to avoid contaminating the liquid medium.

[STEPS]
SECTION: Medium preparation
1. Dissolve 30 g  of   in 
1 L  of  and let mix on the heated magnetic stirrer for 5 min (temperature and mixing speed knob at mid-step).
SECTION: Medium preparation
2. Cover the flask with glass wool and aluminium foil. Autoclave it at 121 °C for 30 min.

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

SECTION: Cultivation of the strains
4. 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. 
SECTION: Cultivation of the strains
5. 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.
SECTION: Preliminary steps
6. Preheat the elution buffer to 70 °C 
SECTION: Preliminary steps
7. 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 500 µL of   to the primary tube.

SECTION: Preliminary steps
8. Cool this tube to -20 °C  on the ice block for 1 min. Then, heat this tube in a water bath at 70 °C for 1 min. Finally, run this tube through the cell disruptor at maximum speed for 1 min. 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.
SECTION: DNA extraction
9. Take 200 µL of the primary lysate (from the preliminary steps) and transfer to a clean tube. Add 25 µL  of proteinase K and 200 µL of lysis buffer BQ1. Homogenize with a vortex for 15 s and then incubate the mixture for 15 min at 70 °C (in the water bath). 
SECTION: DNA extraction
10. Add 200 µL of absolute ethanol (96-100%), vortex for 15 s  and then short-spin centrifuge for MAXIMUM 10 s at    to accelerate protein precipitation. Do not centrifuge any longer. This may cause the DNA to be lost from the supernatant in the pellet.

SECTION: DNA extraction
11. Gently collect the supernatant of the solution (approximately 600 µL) then apply it in the chromatographic column with the silica membrane.
SECTION: DNA purification
12. Centrifuge the column at    for 1 min 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.
SECTION: DNA purification
13. Place the column in a new collection tube and add 350 µL of Buffer BQ2.Then centrifuge at    for 3 min 
SECTION: DNA purification
14. Place the column in a new collection tube (or discard the flows-through) and add 200 µL  of Buffer BQ2. Then centrifuge again at   for 1 min 
SECTION: DNA purification
15. Discard the flows-through or change the collection tube and centrifuge the column at    for 1 min  without adding any buffer.
SECTION: DNA elution
16. Place the column in a clean collection tube. Add 50 µL of elution buffer pre-heated at 70°C to the dried column.

SECTION: DNA elution
17. Incubate for 5 min  at room temperature and then centrifuge for 1 min at   Discard the column and keep the flows-though which is the purified DNA. Store DNA at -80 °C to ensure stability.
SECTION: Spectrophotometric dosage
18. 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.
SECTION: Spectrophotometric dosage
19. 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 2 µL  of the negative control and click on the "blank" box.

SECTION: Spectrophotometric dosage
20. 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.