Mycoplasma pneumoniae Chlamydophila pneumoniae - Mikrogen

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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
II . Sample Extraction/DNA Isolation
Diagenode suggests some appropriate extraction methods:
> Nuclisens easyMAG ® System (Biomerieux)
Clinical samples: nasal swab, whole sputum, bronchoalveolar lavage samples.
Follow manufacturer’s instructions with the following:
Human sample extracted volume: 250 μl (+ 10 μl of the IC DNA virus culture)
DNA isolation volume: 60 μl
The following pre-extraction procedure is recommended:
Bronchoalveolar lavage (BAL):
• 250 μl whole sputum
• Add 250 μl proteinase K buffer (1mg/ml proteinase K, 0,5% SDS, 20 mM Tris-
HCl, pH 8.3), vortex
NB:
Proteinase K (100 mg), Merck, ref: 1.24568.0100
• Incubate 60 minutes at 55°C
• Use the complete 500 μl manipulated sputum for downstream extraction
procedure (Nuclisens Lysis Buffer)
> MagNa pure LC system, Roche: MagNa pure LC Total Nucleic Acid Isolation Kit
(Ref. 03038505001)
Clinical samples: nasal swab, whole sputum, bronchoalveolar lavage samples.
Follow manufacturer’s instructions with the following:
Human sample extracted volume: 200 μl (+ 10 μl of the IC DNA virus culture)
DNA elution volume: 100 μl
The following pre-extraction procedure is recommended:
Bronchoalveolar lavage (BAL):
• Add 3 volume of sputolysin (1:10) with 1 volume of the BAL sample, vortex
NB:
Proteinase K (100 mg), Merck, ref: 1.24568.0100
Bronchoalveolar lavage (BAL):
• Add 3 volume of sputolysin (1:10) with 1 volume of the BAL sample, vortex
NB:
Sputolysin, Calbiochem, ref: 560000
Before using sputolysin, the solution must be diluted 10X with DNA/RNAse water.
• Incubate 15 minutes at room temperature
• Centrifuge 5 minutes at 1500 rpm
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3 e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info@diagenode.com