Phanta max super fidelity dna polymerase kit

Total RNA was extracted from a living earthworm using the Mollusc RNA kit R6875 purchased from OMEGA Bio-tec, America. Then, reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted according to the protocol of the HiScript III 1st Strand cDNA Synthesis kit (Vazyme, Nanjing, China) with the oligo (dT)₂₀ primer. Upon the nucleotide sequence of the transcriptome of P. vulgaris, six pairs of primers (Table 6) were designed to be used in PCR amplification with cDNA by the Phanta Max Super-Fidelity DNA Polymerase kit (Vazyme, Nanjing, China). PCR was performed with 3 µL cDNA template, 1 µL DNA polymerase, 1 µL dNTP mix, and 2 µL of each primer in a 50 µL reaction mixture. Additionally, the PCR protocol was as follows: a pre-denaturation for 3 min at 95 °C was followed by 35 cycles of 15 s at 95 °C, 15 s at the right temperature (Tm), and 1 min at 72 °C. The PCR product was purified by the Gel/PCR extraction kit (Biomiga, San Diego, CA, USA) and subcloned into a pCE2 TA/Blunt-Zero vector at 37 °C using the 5 min TA/Blunt-Zero Cloning kit (Vazyme, Nanjing, China). The recombinant plasmids were transformed into E. coli DH5α and sequenced. The sequencing results were compared with the sequences in the transcriptome to further verify the validity of the primary sequences of the purified proteins.

ChIP assay was performed according to the manufacturer’s instruction of ChIP Assay Kit (Beyotime, #P2078). Briefly, cell samples were crosslinked in 4% paraformaldehyde, lysed, and chromatins were sheared into 200–800 bp by ultrasonicator and hybridized with anti-FOS antibody (CST, #2250) or control rabbit IgG for 12 h. Subsequently, the binding complexes were immunoprecipitated with protein A/G agarose for 1 h and then washed with low-salt wash buffer, high-salt wash buffer, TE buffer, and elution buffer. The retrieved DNA samples were reverse cross-linked and purified with a DNA purification kit (Tiangen, Beijing, China, #DP214). For PCR assay, 8 pairs of primers were designed for NANOG promoter region, and the assay was performed using Phanta Max Super-Fidelity DNA Polymerase Kit (Vazyme, #P505-01). All primer sequences are presented in Table S4.

The coding sequence of MhSultr3;1a (GenBank accession No.: MZ634458) was amplified from the cDNA of M. hupehensis roots with specific primers, namely, MhSultr3;1a-F and MhSultr3;1a-R (Supplementary Table 2), using a Phanta Max Super-Fidelity DNA Polymerase Kit (Vazyme, Nanjing, China) according to the instructions of the manufacturer. Then, PCR amplification was performed as follows: 5 min at 95°C, 35 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products were checked by sequencing (BGI, Shenzhen, China).
Multiple sequence alignments were performed using DNAman (Lynnon Biosoft, San Ramon, CA, United States). The full-length amino acid sequences of Sultr3;1s from M. domestica, Pyrus bretschneideri, Prunus avium, Prunus persica, Populus trichocarpa, and Arabidopsis (GenBank accession numbers see in Supplementary Table 1) were used for phylogenetic analysis with MEGA7.0 (Kumar et al., 2016 (link)).