Premix taq dna polymerase

Total RNA was isolated from SCs using Triquick Reagent (R1100, Solarbio). The RNA must immediately reverse-transcribe to cDNA under the low temperatures using an Evo M-MLV RT Kit (AG11728, Accurate Biology, Changsha, China) in terms of the manufacturer’s instructions. The coding sequence of yak CAV1 (GenBank ID: NW_005394073.1) was amplified by a polymerase chain reaction (PCR; Bio-Rad, Hercules, CA, USA) using Premix Taq DNA Polymerase (TaKaRa, Terra Bella Ave., Moutain View, CA, USA) and cloned into the p-UC-57 vector. DNA fragments encoding the complete yak CAV1 coding sequence were amplified by PCR using Premix Taq DNA Polymerase. The product was then ligated into the pIRES2-EGFP (p-EGFP) vector to construct the pIRES2-EGFP-CAV1 (p-EGFP-CAV1) expression vector. The flanking EcoR I (R0101V, New England Biolabs, Ipswich, MA, USA) and BamH I (R0136V, New England Biolabs) restriction sites were created and the DNA fragments were verified by agarose gel electrophoresis.

Genomic DNA was extracted from Arabidopsis leaves using the CTAB method following the published protocol^{48 (link)}. To detect mutagenesis at the desired sites, the target regions were amplified with specific primers (see supplementary information for primer sequences) using Premix Taq DNA Polymerase (Takara, Japan) with the following protocol: 94 °C for 10 min (94 °C for 30 s, 60–48 °C for 30 s, 72 °C for 2 min) for 13 cycles with touchdown −1 °C in each cycle (94 °C for 30 s, 52 °C for 30 s, 72 °C for 2 min) for 25 cycles, 72 °C for 10 min, 4 °C to hold. The PCR product was separated in a 1% agarose gel and stained with GelStain (TransGen Biotech, China) to detect chromosomal fragment deletions. Selected PCR products were cloned into the pGEM-T Easy Vector (Promega, USA) for Sanger DNA sequencing.

Oligonucleotides were obtained from TsingKe (Beijing, China). DNase I, restriction endonucleases, E. coli inorganic pyrophosphatase, E. coli Poly(A) Polymerase, RNA 5′ Pyrophosphohydrolase (RppH), T4 DNA ligase, NTPs, dNTPs, and RNA purification kits were from New England BioLabs (Ipswich, MA, United States). RNase inhibitor was from Thermo Fisher Scientific (Waltham, MA, United States). PrimeSTAR Max DNA Polymerase and Premix Taq DNA Polymerase, SMARTScribe and ProtoScript II Reverse Transcriptase are were from TAKARA (Shiga, Japan). DNA purification kit was from Axygen (Union City, CA, United States). Ni-NTA resin was from Qiagen (Hilden, Germany). Preparative Superdex S200 for gel filtration was from GE Healthcare (Chicago, IL, United States). Radiolabeled nucleotides were from PerkinElmer (Waltham, MA, United States). 2′-F-dNTPs were from TriLink (San Diego, CA, United States).

To ensure that the primers could simultaneously amplify the target regions of the 18S rRNA gene of all five Babesia spp. without producing nonspecific bands or primer dimers, both of which would interfere with the interpretation of the results in subsequent qPCR-HRM analysis, we performed a series of gradient PCR assays to determine the optimal annealing temperature for the primers. Briefly, the PCR mix (20 μl) containing the Premix Taq DNA polymerase (10 μl; Takara), DNA template (1 μl), each primer (10 μM/μl) and RNase-free water (7 μl) was prepared, and gradient PCR was then performed using a T100™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). Thereafter, the mixture was heated at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at a gradient temperature of 54 °C to 61 °C for 30 s, extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. After gradient PCR amplification, the products were resolved by electrophoresis in 1.5% (w/v) agarose gels to evaluate primer specificity and amplification efficiency at the different annealing temperatures.

We used a protocol based on a recent publication describing monitoring of fresh water for pathogens for this procedure [11 (link)]. Briefly, extracted DNA samples were amplified using the full length of 16s rRNA gene primers with common primer binding sequences 27f and 1492r, attached to unique 24 bp barcodes and nanopore motor protein tether sequence. PCR was performed with 600 nM of each forward and reverse primer, 25 μL of Premix Taq DNA Polymerase (TakaraBio, Shiga, Japan), and a 10 μL DNA template in a 50 μL reaction. The amplification cycles used the following conditions: 94°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds with final elongation at 72°C for 5 minutes. The amplicons from the PCR step were purified using NucleoSpin Gel and PCR Clean-up (Macherey Nagel, Duren, Germany) following the manufacturer’s protocol. The barcoded amplicon samples were pooled in equimolar ratios, and library preparation and sequencing were conducted using Ligation Sequencing Kit SQK-LSK-109 (Oxford Nanopore Technologies, Oxford, UK) on the MinION (Oxford Nanopore Technologies, Oxford, UK) sequencing platform following the manufacturer’s instructions.

PrimeSTAR® GXL DNA Polymerase, Premix Taq™ DNA Polymerase, and PrimeScript™ RT Master Mix were from TaKaRa. TransIT®-2020 Transfection Reagent was from Mirusbio. Fetal bovine serum was from Cellgro. Horse serum was from Gibco. Anti-phosphotyrosine antibody (4G10) was from Merck/Millipore. Anti-Flag-monoclonal antibody was from GNI. Anti-PTPRR antibody was from Thermo Fisher. Anti-MuSK phosphotyrosine (phospho Y754) antibody was from Abcam. Anti-MuSK antibody and agrin were from R&D. Alexa Fluor 555-conjugated α-BTX was from Promokine.

RNA from separate nuclear and cytoplasmic fractions was isolated using PARIS^TM Kit (Thermo Fisher Scientific). Total RNA was extracted and subjected to reverse transcription using the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan). For mRNA and circRNA, a mixture of random and oligo d(T) primers were employed in reverse transcription. For miR-1301-3p, random primers and 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGG ATACGACGAAGTC-3′were used in the reaction system. gDNA was isolated using a TIANamp Genomic DNA Kit (Tiangen).
The Premix Taq DNA Polymerase (TaKaRa) was used for PCR. The cDNA and gDNA PCR products were analyzed using 2.5% agarose gel electrophoresis. Quantitative real-time PCR was performed using the SYBR Premix Ex Taq II (TaKaRa). Relative mRNA and circRNA levels were normalized to GAPDH. In the control group, the gene expression was calibrated as 1. For miRNAs, U6 was used as the internal control. The primers used in PCR and qPCR analyses are summarized in Supplementary Table S2. All the primers span multiple introns and/or exons, and were validated by melting curve analysis.