Initially, 40 mg of stool were mixed with 360 µL of G2 lysis buffer from EZ1®DNA
Tissue Kit (Qiagen, Hiden, Germany). This was mechanically lysed with tungsten
beads (Qiagen, Hiden, Germany) using FastPrep-24TM 5G Grinder for 40 sec. After
10 min of incubation at 100°C to allow for complete lysis, tubes were
centrifuged at 10,000g for 1 min. Subsequently, 200μL of supernatant was
enzymatically digested using 20μL of proteinase K (20mg/mL, Qiagen) and
incubated overnight at 56°C. DNA was extracted from 200µL of sample using the
EZ1®DNA Tissue Kit on BIOROBOT EZ1 (Qiagen, Hiden, Germany), according to the
manufacturer’s instructions. Elution was performed in 200µL volume, then
aliquoted in individual tubes of pure extracted DNA, dilutions to 1:10 and to
1:100.
The extraction quality and the absence of PCR inhibitors were controlled using
the universal eubacterial qPCR targeting the 16S rRNA bacterial genes [14 (link)] on pure DNA, dilutions to 1:10 and to
1:100. By comparison of the Ct values obtained, the dilution to 1:10 was chosen
for the analysis. DNA tubes were stored at -20°C until use.
Tissue Kit (Qiagen, Hiden, Germany). This was mechanically lysed with tungsten
beads (Qiagen, Hiden, Germany) using FastPrep-24TM 5G Grinder for 40 sec. After
10 min of incubation at 100°C to allow for complete lysis, tubes were
centrifuged at 10,000g for 1 min. Subsequently, 200μL of supernatant was
enzymatically digested using 20μL of proteinase K (20mg/mL, Qiagen) and
incubated overnight at 56°C. DNA was extracted from 200µL of sample using the
EZ1®DNA Tissue Kit on BIOROBOT EZ1 (Qiagen, Hiden, Germany), according to the
manufacturer’s instructions. Elution was performed in 200µL volume, then
aliquoted in individual tubes of pure extracted DNA, dilutions to 1:10 and to
1:100.
The extraction quality and the absence of PCR inhibitors were controlled using
the universal eubacterial qPCR targeting the 16S rRNA bacterial genes [14 (link)] on pure DNA, dilutions to 1:10 and to
1:100. By comparison of the Ct values obtained, the dilution to 1:10 was chosen
for the analysis. DNA tubes were stored at -20°C until use.
