Stepone rt pcr instrument

Copy numbers of the nimA gene in B. fragilis 638R/pIP417 was determined by the ΔΔC_T method using SYBR Green detection, the housekeeping glyceraldehyde-phosphate dehydrogenase (gap) gene as reference and the pIP417 repA and nimA genes as target genes. Copies of the nimA gene in clones of 638R/pIP417 adapted to 2 µg/mL and 256 µg/mL metronidazole, respectively (adaptation to metronidazole having been carried out as described above), were measured using unchallenged B. fragilis 638R/pIP417 as the control. Samples were prepared by the boiling method (Sóki et al., 2013 (link)) and the primers used were as follows: gap: gapd1BF 5’-AGCCATTGTAGCAGCTTTTT-3’ and gapd3BF 5’-GAAAACATCATCCCGTCT-3’, repA₄₁₇: repA417-1 5’-TGAGCAACCAGAAAACTC-3’ and repA417-2 5’-TTTTTGCAGCATCCACAA-3’ and nimA: nimART1 5’-GTTCCTGCCGAGTTTACAAC-3’ and nimART2 5’-GATGGTCGAATCCCTTGTCT-3’. The PCR reactions included 5 μL SYBR Select mastermix (LifeTechnologies), 0.7 μM primers, and 1 μL of template preparations in 10 μL of final volumes in triplicate in 48-well PCR plates. Amplifications (95 °C 10 min; 95 °C 15 s, 57 °C 15 s, 72 °C 30 s, 35x and a melting curve from 72 °C to 95 °C) and analysis was made in the StepOne RT-PCR instrument (LifeTechnologies) and by the accompanying software (StepOne Software 2.1, LifeTechnologies).

DNA was isolated from tail snips and tissue samples using the IBI genomic DNA mini kit (Peosta, IA USA) following manufacturer’s protocol. For the PCR reaction, we used Failsafe PCR system (Epicentre Technologies, Madison WI) with PreMix J for the (mT/mG transgene) and Premix H for Ece1^flox allele genotyping.
The mT/mG transgene and Cre-ERT2 allele were identified by PCR from tail DNA preparations. Ece1 KOMP, Ece1^flox and Ece1^Δ alleles were genotyped by PCR using tail and tissue DNA preparations. The list of primers and PCR setting is summarized in Table 1.
The transgene containing the Cre allele was identified by quantitative Real-time PCR reactions, performed in a StepOne RT-PCR instrument (Life Technologies, Carlsbad, CA, USA) using TaqMan assay (Life Technologies, Carlsbad, CA, USA) following manufacturer’s recommended protocol. The TaqMan copy number assay is Mr00635245_cn (Life Technologies, Carlsbad, CA, USA).