Phanta max buffer

The RNA sample used for RT-PCR cloning of the three TRPA genes was prepared by pooling an equal amount of RNA from each of the six developmental stages. A total of 1 μg of this pooled total RNA sample was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). Then, 1 μL of the resultant cDNA sample was used as the template to RT-PCR-amplify the cDNA sequences of TRPA1, painless, and pyrexia, respectively, in a 25 μL reaction containing 12.5 μL 2 × Phanta Max Buffer, 1 μL Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China), 6.5 μL ddH₂O, and 2.0 μL of the gene-specific forward and reverse primers (10 μM) (Table S1) designed based on the contigs of each gene found in the full-length transcriptome of A. hygrophila [41 (link)]. The amplification conditions of PCR were pre-denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and elongation at 72 °C for 4 min, as well as a final elongation at 72 °C for 5 min. The obtained RT-PCR products of each gene were fractioned on a 1.2% agarose gel, eluted using a MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and cloned into pMD™19-T Vector (TaKaRa, Dalian, China). Three positive clones for each gene were sequenced (Sangon Biotech Co., Ltd., Shanghai, China).

The selected single primers of the plant telomere repeat sequence (Table 1) were amplified by single primer PCR using the genomic DNA of G.hirsutum as template, to find the suitable conditions for obtaining promising products and candidates for subtelomeric regions. The amplification procedure was as 95 °C for 3 min, followed by 35 cycles of 95 °C for 15 s, 55 °C/60 °C for 15 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. The amplification products were detected by 1% agarose gel electrophoresis, and the appropriate single primer and annealing temperature were selected based on the above result. Then, PCR amplification was performed using the selected single primer in a 50 μl reaction volume containing 25 μl of 2 × Phanta Max Buffer, 1 μl of Phanta Max Super-Fidelity DNA Polymerase (Vazyme), 0.8 μmol/L of the telomeric single primer, and 10 ng of genomic DNA. The objective band from PCR was recovered by gel extraction kit (SanPrep Column DNA Gel Extraction kit, Sangon Biotech) and was cloned into Trans1-T1 competent cells by the pEasy-Blunt Simple Cloning Vector (TransGen Biotech) according to the manufacturer´s instructions. The positive clones were selected for sequencing by Shanghai Sangon.