Minibest agarose gel dna extraction kit ver 4

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).

PCR products were purified with the MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).

PCR amplification was performed with DTMUV, DuCV, NDRV cDNA, or DNA as a template using three pairs of specific primers, and the PCR products were purified by applying the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Dalian, China) and ligated into the pMD™18-T Vector (China); the constructs were transformed into E. coli DH5α competent cells, coated and incubated at 37°C for 16 h, and the TaKaRa MiniBEST Plasmid Purification Kit Ver. 4.0 (Dalian, China) was applied for extraction of the plasmids, which were named p-DTMUV, p-DuCV, and p-NDRV, respectively, and sequencing. The concentration of each plasmid was determined and normalized using the following formula: plasmid copy number (copies/μL) = plasmid concentration × 10⁻⁹ × 6.02 × 10²³)/(660 Dalton/bases × DNA length).

The M. longifolia used to extract RNA was introduced from the Botanical Garden Berlin-Dahlem in Germany with the accession number of ES-0-B-0180887 and then cultivated at the Germplasm Nursery in the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, Jiangsu Province. Total RNA of M. longifolia leaves was extracted using a FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. After quality and concentration detection, 1 μg of total RNA was used to synthesize the first strand cDNA with a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). To identify the candidate limonene synthase in M. longifolia genome sequence, limonene synthases of M. spicata (AAC37366.1) and M. piperita (ABW86881.1) were used as queries to BLAST in M. longifolia TPSs. Polymerase chain reaction (PCR) was performed to amplify MlongTPS29 with a gene-specific forward primer (5′-ATGGCTTTCAAAGTGTTTAGTG-3′) and reverse primer (5′-TCATGCAAAGGGCTCGAAT-3′). The amplified fragments were purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Takara, Dalian, China) and then cloned into the pClone007 Blunt Simple Vector (Tsingke, Beijing, China). The positive clones were screened and sequenced for confirmation.

The total RNA was extracted and reverse transcription was performed as described above. The primers were designed with Primer Premier 6.0 software according to the DREB1A gene sequence in walnut (Table S1). Amplification was performed using a Biometra TAdvanced 96G PCR instrument (Analytikjena Company, Jena City, Thuringia, Germany). The PCR amplification system followed the manufacturer’s instructions (PrimeSTAR GXL Premix (TaKaRa, Dalian, China)) which contained 12.5 µL of PrimeSTAR GXL Premix, 0.5 µL each of forward and reverse primers (10 µM), 1 µL of cDNA templates and ddH₂O added to 25 µL. The PCR cycling conditions were: 98 °C for 5 min followed by 30 cycles of 98 °C for 10 s, 56 °C for 15 s, 72 °C for 60 s. The amplified products were determined by 1% agarose gel electrophoresis, then purified and recycled using MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), connected with pMD19-T vector (TaKaRa, Dalian, China), and transformed by the Escherichia coli DH5 α competent cell. The full-length cDNA sequence of JfDREB1A gene was obtained by sequencing.

MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0, MiniBEST Viral RNA/DNA Extraction Kit Ver. 5.0, MiniBEST Plasmid Purification Kit Ver. 4.0, EXTAQ, MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0, and pMD ^TM 18-T Vector Cloning Kit were purchased from TAKARA Biomedical Technology (Beijing, China). Syto9 was purchased from ThermoFisher Scientific (Waltham, MA, USA).