Mytaq hs red dna polymerase

Bacterial isolates identified by MALDI-TOF MS with a score of <1.70 were identified with 16S rRNA sequence analysis. Genomic DNA was extracted from pure cultures with the NucleoSpin^® Microbial DNA kit (Macherey-Nagel, Düren, Germany). Extracted DNA was amplified with universal primers BSF 8/20 (5′-AGAGTTTGATCCTGGCTCAG-3′) and BSR 1541/20 (5′-AAGGAGGTGATCCAGCCGCA-3′) with My Taq™ HS Red DNA Polymerase (Bioline, London, UK). PCR was conducted in Mastercycler^® Gradient (Eppendorf, Germany) with following conditions: 95 °C for 1 min followed by 30 cycles at 95 °C for 20 s, 56 °C for 15 s, and 72 °C for 40 s with a final extension at 72 °C for 4 min.
PCR products were sequenced at Microsynt Seqlab (Germany). Sequences were analysed with Geneious (v10.2.6) (Biomatters, Auckland, New Zealand). Consensus sequences (approximately 250 bp) were used in a BLAST search against the NCBI nucleotide database [24 ]. A cut-off value of 99.0% for species and 97.5% for the genus was used in identification.

DNA was extracted for sequencing of the Carnivore protoparvovirus 1 VP2 gene from tissue using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) or from faecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) or by homogenisation, boiling and centrifugation, as previously described [11 (link)]. The extracted DNA was amplified by conventional PCR using 5 u/µL of My Taq HS Red DNA polymerase (Bioline, USA), 1–90 ng of DNA, 1 x MyTaq Red reaction buffer and a final primer concentration of 0.2 µM in a final reaction volume of 25 µL. Three sets of overlapping primers were used to amplify the entire VP2 region (1931 bp) as described previously, with minor modifications (Table 1) [12 (link)]. An initial denaturation step at 94 °C for 1 min, followed by 35–40 cycles of denaturation at 94 °C for 30 s, annealing at 50 or 55 °C for 30 s and extension at 72 °C for 30 s, ending with a final extension at 72 °C for 1 min, was used for DNA amplification. The PCR products were separated by electrophoresis on a 1% agarose gel (Bio-Rad Laboratories, Hercules, CA, USA) in 1× tris-acetate EDTA and visualized using SYBR Safe DNA (Invitrogen, Carlsbad, CA, USA). Sanger sequencing of positive PCR products was performed commercially (Macrogen, Seoul, Korea).

Genomic DNA was extracted using the Extract-N-Amp™ Plant PCR Kit (Sigma-Aldrich) according to manufacturer’s instructions. Presence of the 35S-OsGGP transfer DNA (T-DNA) was determined via multiplex PCR amplification of hptII with OsACT1. The PCR amplification cycles consisted of 1 cycle = 1 min 95°C; 40 cycles = 20 s 95°C, 20 s 60°C, and 90 s 72°C. The PCRs were performed in a final volume of 20 μl for MyTaq™ HS Red DNA Polymerase (Bioline, United Kingdom) according to manufacturer’s instructions.