Mytaq dna polymerase kit

The 5'-UTR region of HCV was amplified by nested PCR using the MyTaq DNA Polymerase kit (catalog number: BIO-21107, Bioline, London, UK). The KY78 and KY80 primers were used for the first amplification and the FIP and RIP primers for the second amplification, as described by Choo et al. [20 (link)] and certified by Holland et al. [21 (link)]. PCR conditions were as follows: initial denaturation at 95 °C for 1 min, followed by 28 cycles of 95 °C for 15 s, 55 °C for 20 s, and 72 °C for 15 s, and a final step of 72 °C for 10 min. For the second PCR, the program was the same, except for using 2 μL from the first PCR as the template. The test was considered positive if a 214-bp product was amplified. OCI was defined as the presence of HCV-RNA in PBMCs, with undetectable levels of anti-HCV and HCV-RNA in plasma. The β-actin gene was also amplified as a PCR control for plasma and PMBC samples of each donor, using the same reagents. The primers sequences were 5'-ATGTGGCCGAGGACTTTGATT-3' (forward primer) and 5'-AGTGGGGTGGCTTTTAGGATG-3' (reverse primer). Amplification was performed under the following conditions: initial denaturation at 95 °C for 1 min, followed by 30 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 15 s, and a final step of 72 °C for 5 min. A PCR product of 110-bp indicated proper RNA isolation and PCR analysis.

Nuclear and cytoplasmic RNA were extracted from E14.5 genital tubercles using the Cytoplasmic and Nuclear RNA Purification kit (Norgen Biotek Corp. ON, Canada). Tissues were frozen in liquid nitrogen and ground using mortar and pestle. RNA was extracted following the manufacturer’s instructions. RNA was eluted in 40 μl elution buffer and yield was approximately 200 ng/μl for cytoplasmic RNA fraction and 40 ng/μl for nuclear RNA fraction. RNA concentrations were determined using NanoDrop ND-1000 Spectrophotometer. cDNA was prepared from 200 ng of RNA using Random Primers and the SuperScript III First Strand Synthesis System (Invitrogen, Sydney, Australia). cDNA was amplified with MyTaq^™ DNA Polymerase kit (Bioline) using primers, PCR-F and PCR-R detecting both Leat1a and Leat1b isoforms.

DNA was extracted from pure microbial cultures using a Chemagic Plant DNA kit with a preceding bead-beating step for mechanical cell disruption (20 (link)). DNA amplification of the 16S rRNA gene using primers 27F and 1492R, as well as Sanger sequencing, was done at commercial sequencing partner LGC Genomics (GmbH, Berlin, Germany). Amplification was performed using the MyTaq DNA Polymerase Kit (Bioline) and Biostab (PCR Optimizer; Bitop AG). PCR quality control was done via agarose gel electrophoresis, followed by ExoSAP-Purification. Sequencing was performed with BigDye Terminator v3.1 (Thermo Life Technologies) on a 3730xl DNA Analyzer. The sequences were blasted against the NCBI sequence database (16S ribosomal RNA) (Bacteria and Archaea type strains, accessed 14 July 2022) for the identification of database entries with highly similar 16S rRNA gene sequences. Sequence alignment including isolates and reference sequences and analysis of evolutionary relationships of taxa were performed in MEGA X (21 (link)). The evolutionary history was inferred using the neighbor-joining method, and the optimal tree (500 replicates in the bootstrap test) was calculated. The evolutionary distances were computed using the p-distance. All ambiguous positions were removed for each sequence pair (pairwise deletion option).

Analysis of differential gene expression between treated and untreated cells was performed using semiquantitative RT-PCR in which 0.5 μg of total RNA was reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) facilitated by random primers in a final volume of 20 μL, according to the manufacturer's protocol. The PCR reaction was performed using 1 μL cDNA template with the MyTaq DNA Polymerase Kit (Bioline) with GAPDH serving as an endogenous control. With the exception of cycle numbers and annealing temperatures (Table S1), PCR conditions were as follows among all tested genes: 94°C for 3 minutes, followed by cycles of 94°C for 30 seconds, annealing temperature for 30 seconds, and 72°C for 45 seconds with a final extension carried out at 72°C for 7 minutes. PCR products were analyzed on 2% agarose gel, and visualization was done using the Gel Doc EZ System (Bio-Rad, USA). Working solution concentrations for all primers used were set to 10 pmol with the exception of GAPDH primers whose working concentration was set to 5 pmol.

To determine genotypes of OVE442 mice, tail tip (fetus and neonates) or ear notches (adults) were used for genomic DNA extraction using DNeasy Blood and Tissue kit (Qiagen, Sydney, Australia). The genomic DNA sequences were amplified with MyTaq^™ DNA Polymerase kit (Bioline, Sydney, Australia) using multiplex primers, OVEMutFwd, OVEMutRev, OVEWTFwd and OVEWTRev detecting wild type and mutant DNA (Supplementary table 1). Amplicons were sequenced by the CTP Sanger Sequencing Service in the Department of Pathology, The University of Melbourne.

We used 5ng of DNA extract for amplification of the barcoding region of the cytochrome c oxidase subunit I gene (CO1). For PCR amplification, we used the MyTaq DNA Polymerase kit (Bioline, Luckenwalde–Germany). For each reaction, 1.5U of MyTaq were pre-mixed with 20μL MyTaq buffer containing 15pmol of the forward and reverse primer, 2μL BioStabII PCR Enhancer (Sigma Aldrich, St. Louis–United States) and 1μL DMSO. We used four different amplicons targeting the CO1 gene (Table 2). For PCR of each DNA extract sample, a unique 8 base-barcode tag was used in the forward and reverse primer. DNA concentrations of amplified amplicons were checked via agarose gel electrophoresis. Approximately 20ng of amplified PCR product of each sample were transferred into amplicon-pools of up to 48 parallel samples. Samples yielding a lower amplicon concentration were amplified for another 5 cycles in an additional PCR reaction.