Gradient thermocycler

The primer pair CR1-CR2 (Sanna et al., 2015) was used to amplify the mtDNA D-loop fragment. A standard 50 μL PCR mixture was used, including 200 ng DNA template, 2.5 mM MgCl₂, 0.2 0 mM each dNTP, 0.20 μM each primer, 0.02 mM BSA, 1 × PCR buffer and 2 units Taq DNA Polymerase (Sigma-Aldrich), according to Mereu et al.^{32 (link)}. PCR amplifications were performed in a Gradient Thermocycler (Eppendorf) by an initial denaturation of 95 °C for 3 min, followed by 30 cycles of 95 °C for 50 s, 60 °C for 30 s, and 72 °C for 1 min.
PCR products were sequenced using the same primers on an ABI 3130 Genetic Analyzer (Applied Biosystem). Sequencing reactions were carried out following the manufacturer's recommendations (BigDye Terminator 3.1 Cycle Sequencing Kit—Applied Biosystem) and purified through the SigmaSpin Post—Reaction Clean—UP Columns (Sigma-Aldrich).
Raw sequencing data were processed by means of the KB base-calling algorithm implemented in the Sequencing Analysis Software 5.3.1 (Applied Biosystem).

To do PCR testing, primers that were designed by De Graaf et al. [19 ] based on the sequence of the 16S rDNA gene of the bacterium were used. Primer sequences were determined based on regions that contain one base difference between P. larvae subsp. larvae and P. larvae subsp. pulvifaciens (AY 030080) at the 3’ ends of the sequences. The expected amplification fragment size was about 700 bp.
F: (5’-TCAGTTATAGGCCAGAAAGC-3’),
R: (5’-CGAGCGGACCTTGTGTTTCC-3’).
The PCR reaction was performed with a final volume of 25 µl, 2.5 µl, 10×PCR buffer, 0.5 µl of 10 mM dNTP mix solution, 1 µl of a concentration of 10 µm of each primer, Taq (1U), 2 µl of 25 mM MgCl₂ solution, and 1µl of extracted DNA and distilled water was used. PCR was performed in an Eppendorf gradient thermocycler with the condition of initial denaturation at 95°C for 1 min and the next 30 cycles as denaturation at 95°C for 1 min, annealing at 55°C for 30s, extending with a temperature of 72°C for 1 min, and a final extending cycle at a temperature of 72°C for 5 min [20 ]. 10µl of PCR product was mixed with 2µl of buffer loading solution and added to 0.8% agarose gel wells containing ethidium bromide. For this, 1 kb of DNA marker was used. After the electrophoresis time was completed, the gel was put on the UV-trans illuminator device to study and take pictures [21 ].

TK6 cells were treated with additional PKC-activating tumor promoters as described previously for TPA. These compounds included PDBu (100 nM), Sapinotoxin D (100 nM), mezerein (100 nM), Ind-V (1000nM) and ROPA (1000 nM). The criteria for dose selection was described previously [13 (link)]. RNA was collected at 8-hours post UVC-irradiation. QPCR was run on an ABI 7500 Real-Time PCR system using ABI master mix and TaqMan® primers (Life Technologies). cDNA was generated with the SuperScript® VILO™ cDNA Synthesis Kit (Life Technologies) with a gradient thermocycler (Eppendorf, Hamburg, Germany). Gene expression levels were calculated using a standard curve for relative quantification with 3 biological replicates per sample. 18S rRNA was used as the housekeeping gene for normalization purposes. Differential expression was determined by comparing against the vehicle treated, non-irradiated samples.