Total DNA was extracted from the frozen fecal samples using a modification of the Qiagen stool procedure and the Qiamp® DNA Stool Mini Kit (Qiagen, Courtaboeuf, France)16 (link). The inner part of the fecal bulk was used for extraction to avoid as much as possible an eventual contamination with soil organisms and/or environmental species during collection as previously described by14 (link). Aliquots of 200 mg of this part were added into tubes containing a 200 mg mixture of 0.1, 0.5, and 2 mm zirconium beads and 1.5 ml of ASL buffer (Qiagen). The samples were mixed vigorously by agitation in a FastPrep BIO 101 agitator (Qbiogene, Strasbourg, France) at 3,200 rpm for 90 seconds. Agitation was followed by heating at 95°C for 10 min to increase both the yield of DNA and proteinase K digestion before the DNA was bound to a column, washed, and eluted in TE buffer.
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Fecal DNA Extraction Protocol
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Corresponding Organization :
Other organizations : Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Université de Montpellier, Institut de Recherche pour le Développement, Méditerranée Infection Foundation
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Variable analysis
independent variables
- Extraction method (modification of the Qiagen stool procedure and the Qiamp® DNA Stool Mini Kit)
- DNA yield
- Use of the inner part of the fecal bulk to avoid contamination with soil organisms and/or environmental species
- Addition of a 200 mg mixture of 0.1, 0.5, and 2 mm zirconium beads
- Addition of 1.5 ml of ASL buffer (Qiagen)
- Mixing by agitation in a FastPrep BIO 101 agitator at 3,200 rpm for 90 seconds
- Heating at 95°C for 10 min to increase both the yield of DNA and proteinase K digestion
- DNA binding to a column, washing, and elution in TE buffer
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