Premix taq

The WSP locus was amplified using the wsp primers (wsp_F1: 5′-GTCCAATARSTGATGARGAAAC-3′ and wsp_R1: 5′-CYGCACCAAYAGYRCTRTAAA-3′) to confirm single infections. The PCR reactions were performed in 25-μl final volumes containing 9.7 μl of ddH₂O, 12.5 μl of Premix Taq (LA Taq Version 2.0, TaKaRa, Dalian, China), 0.8 μl of DNA template, and 1 μl of each primer (10 μM). The thermal cycling conditions were as follows: 95°C for 3 min; 35 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 90 s; 72°C for 10 min. The products were stored at 4°C.
The MLST loci were amplified in accordance with previously published protocols (see text footnote 1). The PCR reactions were performed in 25-μl final volumes containing 9.7 μl of ddH₂O, 12.5 μl of Premix Taq (LA Taq Version 2.0, TaKaRa), 0.8 μl of DNA template, and 1 μl of each primer (10 μM). The thermal cycling conditions were as follows: 94°C for 4 min; 35 cycles of 94°C for 30 s, appropriate T_m (Table 2) for 45 s and 72°C for 90 s; 72°C for 10 min. The products were stored at 4°C.
Each sample was mixed with the same volume of 1× loading buffer containing SYBR green and then separated by electrophoresis on 1.2% (v/v) agarose gels for amplicon detection. All the PCR products were sequenced by Sangon Biotech (Shanghai, China). The sequences of all products were used in BLAST or MLST analyses at the GenBank and PubMLST databases, respectively.

Genomic DNA was extracted from each isolate using a DNA extraction kit (Tiangen, Beijing) according to the manufacturers’ instructions. The virulence genes for ctxAB, ompU, ace, zot, toxR, rtxC, and CTX phage rstR (Classical/El Tor) and tcpA (Classical/El Tor) were amplified using Taq premix (TaKaRa, Japan), the primers and amplification procedures were as described previously (Chow et al., 2001 (link); Singh et al., 2001 (link), 2002 (link); O’Shea et al., 2004b (link)). All of the strains were sequenced for ctxB gene subunit to further identify the characters of the CTX phage, Taq premix (TaKaRa, Japan) was used as described above, and amplification processes were performed as previously described (Goel et al., 2010 (link)). The amplification products were sent for bidirectional sequencing (TaKaRa, Japan), and the results were analyzed using DNAStar (DNASTAR, Inc., United States) and MEGA 4 software (Tamura et al., 2007 (link)). The ctxB sequences of N16961 of El Tor V. cholerae (GenBank: NC-002505) and O395 Classical strain (GenBank: NC-012582) were used as the standards for comparison.

Rabbit aqueous humor was collected before phacoemulsification at 3 days (3D), 1 week (1W), 1 month (1M) after surgery by the 29-gauge insulin syringe (BD Ultra-Fine, Franklin Lakes, NJ, United States). Aqueous humor RNA was extracted with a TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China) and the cDNA was synthesized using the PrimeScript cDNA Synthesis Kit (Takara, Otsu, Japan). Premix Taq (Takara, Otsu, Japan) was used for PCR reaction. The quantitative PCR (qPCR) primers used were: rabbit CSF3 (XM_017349312.1) 5′-CGACTTTGCCACCACCATCT-3′, 5′-GTCAGCTCCAGGAAGCTCTG-3′; rabbit GAPDH (NM_001082253.1) 5′-CGCCTGGAGAAAGCTGCTAA-3′, 5′-CCCCAGCATCGAAGGTAGAG-3′.
The rabbit CSF3 ELISA kit was purchased from Jiangsu Meimian Industrial Co., Ltd. (Yancheng, Jiangsu, China). Rabbit aqueous humor was collected and centrifuged at 3,000 rpm for 15 min to obtain the supernatant. ELISA was performed according to the manufacturer’s instructions.

Total RNA was extracted from human SSC line using RNAiso Plus reagent (Takara, Kusatsu, Japan), and the concentrations and quality of isolated RNA were determined by Nanodrop (Thermo Scientific, Massachusetts, USA). Reverse transcription (RT) was conducted by the HiScript^@II 1^st Strand cDNA Synthesis Kit (Vazyme, R212-02) according to the manufacturer’s instruction. The primers of genes for RT-PCR and real-time PCR were listed in Table 3. The PCR reactions were carried out using Premix Taq™ (TaKaRa Taq™ Version 2.0 plus dye), and the products were analyzed by agarose gels stained with GenGreen nucleic acid gel stain (Genview) and Image Analyzer ChemiDoc XRS⁺ (Bio-Rad).
Real-time PCR reactions were conducted using PowerUp^TM SYBR^® Green Master Mix (Applied Biosystems, Woolston Warrington, UK) and StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The relative expression levels of indicated genes were analyzed via the C_T (threshold cycle) value by normalizing with the housekeeping gene ACTB [ΔC_T=C_T(GENE)−C_T(ACTB)], and the changes of gene expression after treatment compared to the control was calculated by formula 2^−ΔΔCT [ΔΔC_T =ΔC_T(treated)−ΔC_T(control)].

PCR was performed using OPA-02 (5’-TGCCGAGCTG-3’). The composition of the PCR mixture was as follows: 5 µL Premix Taq™ (TaKaRa Taq™ Version 2.0), 5 ng DNA, and 5 µM primers, scaled to 10 µL with PCR-grade water. The PCR was performed according to a previously reported method [12 (link)]. The PCR products were confirmed using the MCE-202 MultiNA capillary electrophoresis system. All amplification reactions were independently repeated at least thrice to obtain reproducible, accurate, and distinct banding patterns. Genotypes were scored as “0” or “1” depending on the absence or presence of bands, respectively. Phylogenetic trees were constructed using the statistical analysis software “R” (version 3.4.3), based on the unweighted pair group method with arithmetic mean (UPGMA method).

cDNA was synthesized using the First Strand cDNA synthesis kit (Sigma-Aldrich) according to the manufacturer’s instructions. PCR was performed by using Premix Taq™ (TAKARA) according to the manufacturer’s instructions. Primers used for the Bmi1 gene were 5′-GAGACCAGCAAGTATTGTCC and 5′-TCTTCATCTGCAACCTCTCC. Primers used for the p16INK4a gene were 5′-GAAGGTCCCTCAGACATCCCC and 5′-CCCTGTAGGACCTTCGGTGAC. Primers used for the p14ARF gene were 5′-GTTTTCGTGGTTCACATCCC and 5′-ACCAGCGTGTCCAGGAAG. The GAPDH gene was used as an internal control (primers: 5′-TGCCAAATATGATGACATCAAGAA and 5′-GGAGTGGGTGTCGCTGTTG). PCR was performed using an initial denaturation at 94 °C for 5 min, followed by 18–25 (GAPDH: 18 cycles, Bmi1: 22 cycles, p16INK4a and p14ARF: 25 cycles) cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 45 s. After amplification, an additional elongation step was performed at 72 °C for 10 min. Amplified PCR products were run on 1.5% agarose gels containing 0.5 g/ml ethidium bromide.