Ex taq dna polymerase kit

Methylation-specific PCR (MS-PCR) was performed to evaluate the methylation status of Sirt1. Firstly, genomic DNA from the cultured cells was isolated using a Universal Genomic DNA Extraction Kit Ver3.0 (Takara Bio). The generated DNA of each sample was experienced bisulfite modification process as follows: degeneration in 3 mol/L NaOH for 15 min at 37°C; sulfonation of cytosines in 3.6 mol/L sodium bisulfite and 1 mmol/L hydroquinone (Sigma–Aldrich) for 16 h at 55°C. Following, modified DNA samples were desalted using a DNA clean-up system (Promega Corporation). After treatment, Sirt1 was amplified using Takara's Ex Taq™ DNA Polymerase kit basing on two primers of Sirt1 which cover almost the entire CpG rich region of the proximal Sirt1 promoter in Biomedical Instrumentation Center (USUHS). Relative level of MS-PCR products were analysed by a 2% agarose gel and normalized to non-methylation 16S RNA.

Cassava 14-3-3 proteins were identified in the phytozome v10 database (using 14-3-3 as the keyword search on the website https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Mesculenta) (Goodstein et al., 2012 (link)). Me14-3-3 genes were amplified based on the search sequences to design primers by using Primer 5.0. These primer sequences are listed in Supplementary Table S4. The PCRs were carried out in a final volume of 50 µL, containing 1 µL of cDNA from different tissues, by following the manufacturer’s instructions of the Ex Taq DNA polymerase kit (Takara, Maebashi, Japan). The PCR cycling conditions were as follows: 3 min at 94°C, followed by 30 cycles of 94°C for 30 s, a range of annealing temperatures for different types of Me14-3-3s from 57°C to 63°C for 30 s, 72°C for 1 min, and a final extension of 10 min at 72°C. The PCR products were separated on 1% agarose gel and purified by an Axygen Purification kit (Axygen, Union City, CA, USA), cloned into pMD19-T vector (Takara, Japan), and sequenced (The Beijing Genomics Institute, Guangzhou, China). All of Me 14-3-3 genes were analyzed by using ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/) software and the physicochemical properties of amino acids were analyzed by using the ProtParam tool (http://www.expasy.ch/tools/protparam.html) software.

Full-length cDNAs of the MeFRK genes were isolated via RT-PCR, using a set of gene-specific primers (Table 2), which were designed based on BLAST analysis of the cassava genome database (Available online: http://www.phytozome.net/cassava) using seven published sequences of the AtFRK1–7 in A. thaliana [11 (link)]. The PCR reaction was carried out at a final volume of 50 μL, containing 1 μL of cDNA from different tissues, following the manufacturer’s instructions from the Ex Taq DNA polymerase kit (Takara, Japan). The PCR cycling conditions were as follows: 3 min at 94 °C, followed by 30 cycles of 94 °C for 30 s, a range of annealing temperatures for different MeFRKs from 57 to 63 °C for 30 s, 72 °C for 2 min, and a final extension of 10 min at 72 °C. The PCR products were separated on 1% agarose gel and purified by Axygen Purification kit (Axygen, Union, CA, USA), cloned into pMD18-T vector (Takara, Dalian, China), and sequenced (Shanghai Sangon Biological Engineering Technology and Services Co., Ltd, Shanghai, China).

The HBoV-positive samples were selected, and the TaKaRa Ex Taq DNA Polymerase kit (Code: RR001A) was used for panbocavirus nested PCR amplification of the 576-bp HBoV VP1/2 gene. The first-round PCR reaction conditions were as follows: 95°C for 2 min, followed by 10 cycles, 95°C for 35 s (58°C for 1 min, minus 0.5°C for each cycle), 72°C for 1 min, followed by 30 cycles, 95°C for 30 s, 54°C for 45 s, and 72°C for 45 s, and finally extended for 10 min at 72°C. Similar conditions were repeated except that the initial annealing temperatures in the first and second groups of PCR cycles in the second round were changed to 60 and 58°C, respectively. The primer sequences used are shown in Table 1 (Kapoor et al., 2010 (link); Lu, 2013 ). The amplified products were then sent to sequencing companies for sequencing.

As described in the Supplementary Methods, viral RNA extracted from the inferred dually infected individuals was reverse-transcribed to gp41 complementary DNA (cDNA) using SuperScript^® IV First-Strand Synthesis System (Invitrogen, Catalog No: 18080-051), and cDNA was endpoint diluted in 96-well plates such that fewer than 29 PCRs yielded a amplification product. According to a Poisson distribution, the cDNA dilution that yields PCR products in no more than 30% of wells contains one amplifiable cDNA template per positive PCR more than 80% of the time (Salazar-Gonzalez et al., 2008 (link)). The diluted gp41 cDNA was used to perform first-round PCR according to the protocol of the ExTaq DNA Polymerase Kit (Takara, RR001B), and the second-round amplification was carried out based on the same conditions as the bulk PCR amplification using the Premix ExTaq Kit (Takara, RR902A). All products derived from cDNA dilutions yielding less than 30% PCR positivity were sequenced.

Total genomic DNA was extracted from the muscle by standard phenol-chloroform procedures [24 ]. The mitogenomes of anemonefishes were determined using eight consensus primer pairs with a long PCR technique (Table 2) [25 (link)]. PCR amplifications were carried out on an ABI 2700 Thermo Cycler (www.appliedbiosystems.com) in 25 μl reaction volumes, by using Takara Ex Taq DNA polymerase kit (www.takara.com) as indicated by the manufacturer. PCR amplifications were under the following standard cycle: one denaturation step at 94°C for 3 min, 30 cycles of 94°C for 45 s, 50~60°C for 1 min and 72°C for 1 min 30 s, followed by a final elongation step at 72°C for 10 min. PCR products were sequenced on an ABI3730XL DNA Analyzer (Sangon Biotech, www.sangon.com)

We amplified the V4 variable region of bacterial 16S rRNA genes from the DNA samples by PCRs (one PCR with one primer set for each DNA sample) using a TaKaRa Ex Taq^® DNA Polymerase Kit (Takara Bio United States, Inc., Mountain View, California, United States). The forward primer 515F (5′-GTG CCA GCM GCC GCG GTAA-3′) was identical for all PCRs. However, each PCR had a unique reverse primer consisting of 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′) and a sample-specific Golay barcode (Caporaso et al., 2012 (link); Walters et al., 2016 (link)). Each PCR totaled 25 μL with 200 nM each (forward and reverse) primer and 2 μL of DNA sample (equivalent to 20 mL of original water sample). The PCR thermal cycling conditions were 5 min at 94°C, 30 cycles of (45 s at 94°C, 60 s at 50°C, and 90 s at 72°C), and 10 min at 72°C (Qin et al., 2017 (link)). After checking the quality of the PCR amplicons with agarose gel electrophoresis, we pooled and purified the amplicons of different DNA samples. The purified amplicons were paired-end sequenced (2 × 250 bp) on an Illumina MiSeq platform (Illumina, Inc., San Diego, California, United States) at the DNA Core Facility of Cincinnati Children’s Hospital (Cincinnati, Ohio, United States).