Mastercycler gradient pcr machine

K. pneumonia subsp. pneumonia was confirmed based on 16S–23S rDNA internal transcribed spacer (ITS) using specific primers (described in Table 1). Polymerase chain reaction (PCR) condition was as previously described.
¹⁷ (link) String test was used for phenotypic identification of hvKP. An inoculation loop was touched to the surface of the colony on an agar plate. Bacterial isolates that were able to form a viscous string >5 mm in length were confirmed as hvKP. For genotypic confirmation of hvKp isolates, all string‐positive ones were screened for targeting magA, iucA, and rmpA by PCR analysis.
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The PCR reaction mixture was prepared in a final volume of 20 µL contained 12.5 µL of 2× Master mix (Amplicon), 0.5 μL of each primer (10 pM/μL), 2 µL of DNA template, and 4.5 μL of distilled water. The microtubes containing reaction mixture were transferred in the Master Cycler gradient PCR Machine (Eppendorf) and PCR was done as follows: initial denaturation at 94°C for 4 min; 35 thermal cycles of denaturation at 94°C for 45 s, annealing at 57–58°C and 61°C for ITS, magA, iucA, and rmpA, respectively (for 30 s), and extension at 72°C for 45 s; and a post‐PCR final incubation at 72°C for 5 min. The amplified PCR products were electrophoresed on 1.5% agarose gel containing 0.5 μg/mL safety stain and visualized under a UV transilluminator.

Human total RNA from cardiac and skeletal muscles was obtained from a human total RNA Master Panel II (Clontech Laboratories, Inc, Mountain View, CA). cDNA was synthesized from 0.5 µg of each total RNA using random primers as described.^{22 (link)} The cDNA fragment extending from exon 1 to exon 8 was PCR amplified using the primer set; 1c (5′-ATGCTTTGGTGGGAAGAAGTAG-3′) and c8r (5′-GTAGGACTTCTTATCTGGATA-3′), and the cDNA fragment extending from the Dp116 specific exons 1 to 62 was PCR amplified using the primer set; Dp116ex1F (5′-GGGTTTTCTCAGGATTGCTATGC-3′) and 4F (5′-GAGGAGGTCAATACTGAGTGGG-3′). Amplifications were performed in a total volume of 10 µL, containing 1 µL cDNA, 1 µL 10× ExTaq buffer (Takara Bio, Inc, Shiga, Japan), 0.25 U ExTaq polymerase (Takara Bio, Inc), 500 nmol/L of each primer, and 250 µmol/L dNTPs (Takara Bio, Inc) on a Mastercycler Gradient PCR machine (Eppendorf AG, Hamburg, Germany). The amplification protocol consisted of an initial denaturation at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 3 minutes. Amplified PCR products were electrophoresed using a DNA 1000 LabChip kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Reverse transcription-PCR of the GAPDH gene (glyceraldehyde 3-phosphate dehydrogenase) was performed as described.^{23 (link)}