Genescan 600 liz

Bacterial community structures were monitored by amplifying the hypervariable regions V1–V3 of the 16S rRNA gene with the general bacterial primers fD1 and 5′FAM-labeled PRUN518r (Mikkonen et al. 2011 (link)). The volume of one PCR reaction was 50 μL, containing 2 μL undiluted DNA extract, 0.2 mM of each dNTP (Thermo Scientific), 0.3 μM of each primer (Oligomer), 0.7 mg mL⁻¹ BSA (Thermo Scientific), 1× PCR buffer (Biotools), and 1 U DNA polymerase (Biotools). The amplification was done in a DNA Engine DYAD^™ thermal cycler (MJ Research Inc., USA) with the following program: 5 min at 95 °C followed by 28 or 30 cycles of 45 s at 94 °C, 1 min at 55 °C, 1 min at 72 °C, and finally 5 min at 72 °C. The PCR products were checked by agarose gel electrophoresis (1.5 % agarose gel run at 100 V for 1 h). Fragment analysis by polyacrylamide capillary electrophoresis (ABI 3130 XL, Applied Biosystems) was outsourced to DNA Sequencing and Genomics Laboratory Core Facility, University of Helsinki, Finland, using GeneScan 600 LIZ (Applied Biosystems) as sizing standard.

The genetic assessment of the present study employed the method reported by Bonassi et al. [75] (link). DNA extraction and genotyping were performed by ACGT, Inc. (Wheeling, IL). DNA was extracted by using Oragene DNA purification reagent and its concentrations were evaluated through spectroscopy (NanoDrop Technologies, USA). Each DNA sample was increased by polymerase chain reaction (PCR) for the OXTR gene rs53576 region target with the primers 5-GCC CAC CAT GCT CTC CAC ATC-3 and 5-GCT GGA CTC AGG AGG AAT AGG GAC-3. A PCR reaction of 20 ll, comprising 1.5 ll of genomic DNA from the test sample, PCR buffer, 1 mM each of the forward and reverse primers, 10 mM deoxyribonucleotides, KapaTaq polymerase, and 50 mM MgCl2 was executed. PCR operation comprised of 15 minute denaturation at 95 ^∘C, and 35 cycles at 94 ^∘C (30 s), 60 ^∘C (60 s), 72 ^∘C (60 s) and a final 10 minute step at 72 ^∘C. PCR reactions were genotyped with an ABI 3730xl Genetic Analyzer (Applied Biosystems Inc.) and normalized with GeneScan 600 LIZ (Applied Biosystems, Inc.) size standards on each sample. Genotypic data were inspected using GeneMapper ID (Applied Biosystems, Inc.).
The same procedure was used to assess the OXTR gene rs2254298 region. However, the forward and reverse primers were instead 5-TGA AAG CAG AGG TTG TGT GGA CAG G-3 and 5-AAC GCC CAC CCC AGT TTC TTC-3 respectively.