May 15, 2022
DNA extraction from dermatophytes using the Macherey-Nagel NucleoSpin™ Blood QuickPure kit (REF: 740569)
- 1AIDS Reference Laboratory, Department of Clinical Microbiology, University Hospital of Liege, 4000 Liege, Belgium
Protocol Citation: Khalid El Moussaoui 2022. DNA extraction from dermatophytes using the Macherey-Nagel NucleoSpin™ Blood QuickPure kit (REF: 740569). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwkbpzvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: March 25, 2022
Last Modified: May 15, 2022
Protocol Integer ID: 59914
Keywords: dermatophytes, dna extraction, nucleospin
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Abstract
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.
Materials
Macherey-Nagel NucleoSpin™ Blood QuickPure kit (REF: MN 740569)
Sabouraud Dextrose Broth (REF : Merck S3306)
Cell Lysis Solution for genomic purification (REF : Promena A7933)
Protocol materials
MilliQ Water
Step 1
Cell Lysis Solution for Genomic PurificationPromegaCatalog #A7933
Step 7
Sabouraud dextrose brothMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3306
Step 1
Medium preparation
Medium preparation
4h 30m
4h 30m
Dissolve 30 g of Sabouraud dextrose brothSigma AldrichCatalog #S3306 in
1 L of MilliQ WaterSigma Aldrich and let mix on the heated magnetic stirrer for 00:05:00 (temperature and mixing speed knob at mid-step).
10m
Cover the flask with glass wool and aluminium foil. Autoclave it at 121 °C for 00:30:00 .
4h
Cultivation of the strains
Cultivation of the strains
4d
4d
After allowing to cool, transfer 25 mL of this medium into a tube. Label the tube with the strain number.
1h
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.
1m
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.
4d
Preliminary steps
Preliminary steps
25m
25m
Preheat the elution buffer to 70 °C
1m
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 Cell Lysis Solution for Genomic PurificationSigma AldrichCatalog #A7933 to the primary tube.
2m
Cool this tube to -20 °C on the ice block for 00:01:00 . Then, heat this tube in a water bath at 70 °C for 00:01:00 . Finally, run this tube through the cell disruptor at maximum speed for 00:01:00 . 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.
20m
DNA extraction
DNA extraction
25m
25m
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 00:00:15 and then incubate the mixture for 00:15:00 at 70 °C (in the water bath).
20m
Add 200 µL of absolute ethanol (96-100%), vortex for 00:00:15 and then short-spin centrifuge for MAXIMUM 00:00:10 at 11000 x g to accelerate protein precipitation. Do not centrifuge any longer. This may cause the DNA to be lost from the supernatant in the pellet.
1m
Gently collect the supernatant of the solution (approximately 600 µL ) then apply it in the chromatographic column with the silica membrane.
1m
DNA purification
DNA purification
6m
6m
Centrifuge the column at 11000 x g for 00:01:00 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.
1m
Place the column in a new collection tube and add 350 µL of Buffer BQ2.Then centrifuge at 11000 x g for 00:03:00
3m
Place the column in a new collection tube (or discard the flows-through) and add 200 µL of Buffer BQ2. Then centrifuge again at 11000 x g for 00:01:00
2m
Discard the flows-through or change the collection tube and centrifuge the column at 11000 x g for 00:01:00 without adding any buffer.
1m
DNA elution
DNA elution
8m
8m
Place the column in a clean collection tube. Add 50 µL of elution buffer pre-heated at 70°C to the dried column.
1m
Incubate for 00:05:00 at room temperature and then centrifuge for 00:01:00 at 11000 x g Discard the column and keep the flows-though which is the purified DNA. Store DNA at -80 °C to ensure stability.
7m
Spectrophotometric dosage
Spectrophotometric dosage
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 2 µL 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.