Rtaq dna polymerase

Total S. alopecuroides DNA was extracted using a DNA extraction kit (Fast DNA^® SPIN Kit, MPBIO, Santa Ana, USA) and DNA amplification experiments were performed using an ABI GeneAmp^® Model 9700 PCR instrument. Fungal primers ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2R (5’-GCTGCGTTCTTCATCGATGC-3’) were used to amplify the ITS1 region of rDNA [37 (link)]. The reaction system was 20 μL: 10×Buffer 2 μL, 2.5 mmol/L dNTPs 2 μL, 5 μmol/L 0.8 μL each for forward and reverse primers, TaKaRa rTaq DNA Polymerase 0.2 μL, BSA 0.2 μL, DNA template 10 ng, and ddH₂O was made up to 20 μL. PCR procedure: 95 °C for 3 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s; 72 °C for 10 min, 10 °C for holding. PCR products were sampled on 2% agar gel electrophoresis in 3 μL for detection.
The purity and concentration of PCR products and DNA integrity, PCR amplification, and Illumina MiSeq sequencing were performed by Majorbio Co., Ltd. (Shanghai, China). All sequencing data were deposited to the NCBI Sequence Read Archive2 under project number PRJNA832900.

Total RNA was extracted using a plant-specific RNA kit (Invitrogen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from RNA isolated from P. trichocarpa using M-MLV reverse transcriptase. Primers (Supplement Table 1) were designed and synthesized for defensin ORF cloning, the PCR system includes 2 µL forward and reverse primers, 2.0 µL cDNA as template, 5.0 µL 10× PCR buffer (Mg²⁺ plus), 1 µL 10 mM dNTPs, 0.5 µL rTaq DNA polymerase (Takara, Japan) and the ddH₂O was to a constant volume up to 50 µL. Also, the PCR reaction was performed as follows: 95 °C for 10 min, 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s and, finally, 72 °C for 10 min. Also the PtDef PCR product was purified (Takara, Japan) and inserted into the PEASY-T3 plasmid (TransGen Biotech, China). Subsequently, the positive clones were sequenced using the M13 primers. The nucleotide sequence, deduced amino acid sequence, and ORF of defensin from P. trichocarpa were analyzed using the ExPASy online package (http://www.expasy.org/translate/). We then calculated the molecular weight (Mw) of defensin and predicted its isoelectric point (pI) using the ExPASy pI/Mw tool (http://www.expasy.org/tools/pi_tool.html).

Genomic DNAs were extracted from the spleen samples using the E.Z.N.A. tissue DNA extraction kit (Omega Bio-tek, Norcross, GA, US) according to the manufacturer’s protocol. The quality and quantity of extracted DNAs were examined by 1% agarose gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, US). The V3-V4 regions of the 16S rDNA were amplified by PCR in a thermal cycler GeneAmp 9700 (Applied Biosystems Inc, Foster City, CA, US). The PCR condition was 95°C for 3 min, followed by 30 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s and a final extension at 72°C for 10 min. The primers used were 338F (5’-barcode-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The barcode is an eight-base sequence unique to each sample. The PCR reactions were performed in triplicate in 20 μL mixture containing 2 μL of 10 × PCR Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.2 μL of rTaq DNA Polymerase (TaKaRa Bio, Dalian, China), and 10 ng of template DNA.

The PCR primers (divergent and convergent) were designed for circRNA validation (Supplemental Table 5). The cDNA retro-transcribed from total RNA treated with DNase I and RNase R and the genomic DNA were used as templates. Convergent primers were used as positive controls for linear transcripts, and divergent primers were used to detect the candidate circular template. For each PCR amplification, 20 ng of cDNA or genomic DNA was used with rTaq DNA polymerase and 10× buffer (Takara). We performed 35 cycles of PCR. To confirm the PCR results, the PCR products of the expected length were dissected from a gel and purified using the QIAquick Gel Extraction Kit (Qiagen). Direct sequencing was performed on an ABI 3730 sequencer according to the manufacturer's protocol.

To determine BMCC1 mRNA expression levels, total RNA was isolated using the RNeasy Mini Kit (Qiagen) and was reverse-transcribed using random primers and SuperScript II reverse transcriptase (Life Technologies). The cDNA was PCR amplified using rTaq DNA polymerase (Takara Bio, Shiga, Japan). PCR primer sequences were used as follows: (forward and reverse): BMCC1-F, 5′-CTCATCACCGAGCAACTGGCTCATC-3′ and BMCC1-R, 5′-CACTGCCTGCCACGGCTTCTGTTG-3′ p21-F, 5′-GCGATGGAACTTCGACTT-3′ and p21-R, 5′-CAGGTCCACATGGTCTTCCT-3′ and GAPDH-F, 5′-ACCACAGTCCATGCCATCAC-3′ and GAPDH-R, 5′-TCCACCACCCTGTTGCTGTA-3′. GAPDH expression served as an internal control.

As shown in Fig 1A, isolated taste buds were collected from the digested epithelial tissues of rat CP by means of a patch pipette using a micromanipulator (NARISHIGE, Tokyo, Japan), and directly reverse-transcribed with a CellAmp whole transcriptome amplification kit (Takara, Shiga, Japan) and Ex Taq Hot start version (Takara) according to the manufacturers’ instruction manuals, and then the cDNA-amplified products were used as templates for isolated taste buds. Total RNA in taste bud-containing epithelium of rat CP was extracted and reverse-transcribed with a NucleoSpin RNA^Ⓡ XS kit (Macherey-Nagel, Düren, Germany), and a PrimeScript^™ RT reagent kit with gDNA Eraser (Takara) according to the manufacturers’ instruction manuals. Nested PCR was performed using rTaq DNA polymerase (Takara), and the primer sets, annealing temperatures, and cycle numbers were shown in Table 1. All reactions for PCR were performed with the following parameters: 94°C for 5 min; the designated cycles of 94°C for 30 s, the designated annealing temperature for 30 s, and 72°C for 30 s; and a final step at 72°C for 10 min. A negative control involving the template (H₂O control) was set in all PCR reactions. The PCR products were confirmed by sequence analysis (data not shown).

Three additional pairs of PCR primers were designed using DNAStar6.0 based on published CAV sequences in GenBank, which were expected to generate amplicons of 843, 989, and 802 bp each in length encompassing the entire CAV genome (Supplementary Table S1). The total DNA was extracted from the cultured cell supernatant of the 24 CAV positive samples. PCR were performed in a 25 μL reaction volume containing 0.5 μL forward primer, 0.5 μL reverse primer, 1 μL DNA, 2.5 μL 10 × PCR buffer (Mg²⁺), 2 μL dNTPs (2.5 mM), 0.5 μL rTaq DNA polymerase (TaKaRa Biotechnology Co. Ltd., Dalian, China), and 18 μL double-distilled H₂O. PCR cycle conditions were 95°C for 5 min for pre-denaturation, followed by 30 cycles of 95°C for 30 s, 55°C for 50 s, and 72°C for 60 s; a final extension step was set at 72°C for 10 min prior to termination of the reaction at 4°C. The PCR products were analyzed by 1% agarose gel electrophoresis.
All PCR for each sample were performed in duplicate. Both positive and negative controls were included for each batch of PCR. The PCR products were analyzed in 1% agarose gels stained with ethidium bromide. The amplicons were purified with a Gel Band Purification Kit (Omega Bio-Tek, USA), cloned into the pMD-18T vector (TaKaRa Bio Inc., Japan), and the sequenced in triplicate on an ABI 3730 Sanger-based genetic analyzer (Carlsbad, CA, USA).