Gel doc xr image system

Immunoblots were conducted following the previously described protocol (Solesio et al., 2012 (link); Solesio et al., 2013a (link); Baltanas et al., 2013 (link)), and using 10 ug of protein per condition. Protein quantification was conducted as indicated above. All primary and secondary antibodies were used at 1:1,000 dilution. The signal was detected using the Gel Doc XR Image System form BioRad (Hercules, California, US). TOMM20 protein levels were used to confirm mitochondrial enrichment in the mitochondrial fractions, calreticulin levels to confirm ER enrichment in the ER fractions, and β-actin levels as loading controls in the rest of the experiments.

All specimens containing M. pneumoniae DNA had been stored at −70 °C before testing. Nucleic acid extraction was performed using the Ribospin TM vRD plus kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. The extracted DNA was subjected to PCR using the Pfu Plus 5× Master mix (Elpis Biotech, Daejeon, Korea) according to the manufacturer’s instructions. The primers targeted residues 1998–2018 (forward) and 2673–2692 (reverse) in domain V of 23S rRNA (Myco23S-F: 5-TCTCGGCTATAGACTCGGTGA-3 and Myco23S-R: 5-TAAGAGGTGTCCTCGCTTCG-3).
The PCR products were subjected to 1% agar gel electrophoresis using a submarine-type electrophoresis device (Mupid-exU; Takara Bio Inc., Shiga, Japan). The approximately 700-bp band was identified under ultraviolet light, photographed using an imaging system (Geldoc XR image system; BioRad, Hercules, CA, USA), and then excised. The gel extraction procedure was performed using a commercial gel extraction SV kit (MGmed, Seoul, Korea), and the product was analyzed using the BigDye^® Terminator v3.1 Cycle Sequencing and an ABI PRISM 3730XL Analyzer (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). The DNA sequences were evaluated for point mutations at residues 2063, 2064, and 2067 in domain V of 23S rRNA using a sequence that is registered in the GenBank database (GenBank number: X68422).

The formation of circular DNA was verified using urea-PAGE electrophoresis. Briefly, 10 μL of DNA circularization product was premixed with 2 μL 6X DNA gel loading dye and then loaded onto premade15% Mini-PROTEANR TBE-Urea Gel (Bio-Rad, 4566055). The electrophoresis was then carried out for 60 min at a constant voltage of 110 V. 2.5 μL of 10 bp DNA ladder was used for molecular weight reference. Gel images were visualized by using Gel Doc + XR image system (Bio-Rad Laboratories Inc., USA).

1 μL of 100 μM LbaCas12a endonuclease (NEB, M0653T) and 5 μL of 20 μM gRNA was mixed with 3.6 mL 1× NEBuffer 2.1, and followed with the adding of 1 μM non-trigger ssDNA or dsDNA oligo. Then, 10 μL of 1 μM trigger ssDNA was mixed with 90 μL of the prepared standard CRISPR/Cas12a reaction mixture. The reaction was set at room temperature for 0–60 mins, and each of 10 μL cleavage product from 0, 10, 20, 30, 40, 50, 60 min was mixed with 2 μL of 6X DNA gel loading dye, and then loaded onto 4% agarose gel for electrophoresis at a constant voltage of 80 V for 60 min. Gel images were visualized by using Gel Doc + XR image system (Bio-Rad Laboratories Inc., USA).

Total RNA was isolated using the Trizol reagent (Gibco-BRL) according to the manufacturer's protocol. Total RNA (1 μg) was reverse-transcribed by a ReverTra Ace-α Kit (TOYOBO) according to the manufacturer's protocol. The cDNA was diluted 10-fold, and 1 μl of diluted cDNA was used in a 20 μl PCR reaction. Semiquantitative RT-PCR was performed using gene-specific primers and actin11 (SfACT11) as the housekeeping gene. PCR was performed with a 5 min denaturation at 94°C, followed by 28 cycles of 94°C for 30 sec each at 55°C, followed by 72°C for 1 min. PCR products were analyzed by using 1.2% agarose gel, stained with ethidium bromide (EtBr), and visualized under ultraviolet light by Gel Doc™ XR+ image system (Bio-Rad). The densitometry data for band intensities was generated by analyzing the gel images using the Image Lab™ Software (Bio-Rad).