Amplitag gold dna polymerase

IAV RNA was isolated using the QIAmp® Viral RNA Mini Kit (Qiagen) following manufacturer’s instructions and reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen) with the Uni12 primer (5’-AGCAAAAGCAGG-3’) (40 (link)). Chimeric NA cDNA regions were amplified using AmpliTag Gold DNA polymerase (Applied Biosystems) with primers NA int for (5´-ATCTGTCTGGTAGTCGGA-3´) and NA int rev (5´-GGCCAAGACCAATCTACA-3´). For amplification of the hemagglutinin (HA) gene segment, primers HA for (5’-AGCAAAAGCAGGGG-3’) and HA rev (5’-AGTAGAAACAAGGGTGTTTT-3’) were used. PCR products were separated on 0.8% agarose TBE gel and sequence identity of NA and HA was confirmed by sequencing (Microsynth Seqlab).
NS1 gene sequence integration into deletion site III of the MVA genome was verified by PCRs specific for the six major deletion sites of MVA as described previously (39 (link)). Purified PCR products were separated on 1% agarose TBE gel and analyzed with imaging system (ChemiDoc, ImageLab v6.0.1, Bio-Rad Laboratories, Inc.). For sequencing of NS1, deletion site III-specific PCR was performed (39 (link)) and purified PCR product was sequenced (Microsynth Seqlab). For PCRs, GoTaq® DNA polymerase (Promega) and for DNA purification, GeneJET Gel Extraction Kit (Thermo Scientific™) were used.

PCR was performed to prove the absence or presence of bacteria in the UCM and to provide amplicons for DGGE. Samples for analysis of the tripartite community and for axenicity tests were taken weekly under sterile conditions. An amount of 50 mL of culture medium was filtered using a polycarbonate filter (Millipore ISOPORE^(TM), 0.2 μm GTTP 25 mm, Sigma Aldrich, München, Germany) in a polysulfone filter holder for syringes, and stored at –80 °C until extraction using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). DNA was extracted based on the protocol of the QIAmp Blood DNA extraction kit (Qiagen Manual, 2nd Edition) metagenomics. All experiments were performed under strictly sterile conditions.
The nucleotide sequence for the forward primer, which is specific for eubacteria, contains a GC clamp (357fGC; CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG). The universal consensus sequence was used as reverse primer (907rM; CCG TCA ATT CMT TTG AGT TT) [53 (link)]. The PCRs, as well as the DGGE, were performed by using AmpliTag Gold DNA Polymerase (Applied Biosystems, Foster City, CA, USA) according to Grueneberg et al. [11 (link)].

Detection of Coronavirus HKU15 was performed by polymerase chain reaction (PCR) amplifying a 289-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene, using the specific primer pair LPW14077 (5′-ACA CAC TTG CTG TAA CCA AA-3′) and LPW14080 (5′-ATC ATT AGA GTC ACC ACG AT-3′). PCR and DNA sequencing were carried out following our previous publications with slight modification.^{26 (link), 27 (link)} Briefly, each PCR mixture contained PCR buffer (50 mM of KCl, 10 mM of Tris-HCl at pH 8.3 and 3 mM of MgCl₂; Applied Biosystems, Foster City, CA, USA), 200 μM of each deoxynucleoside triphosphate (Roche Diagnostics, Basel, Switzerland), 1 μM of each primer (Invitrogen), 1.0 U of AmpliTag Gold DNA polymerase (Applied Biosystems) and cDNA. The mixtures were subjected to 60 thermocycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min, with an initial denaturation at 95 °C for 10 min and a final extension at 72 °C for 10 min for DNA amplification using the GeneAmp PCR System 9700 automated thermal cycler (Applied Biosystems). Standard precautions were taken to avoid contamination, and no false-positive result was observed for the negative controls. PCR products were agarose gel-purified using the QIAquick Gel Extraction kit (Qiagen). Both strands of the PCR products were sequenced twice by the ABI Prism 3130xl Genetic Analyzer (Applied Biosystems) using the two PCR primers.

The PCR mixture contained 10× Gold buffer (Applied Biosystems, USA), 2 μL of 25 mM MgCl₂, 0.5 μL of 10 mM dNTPs, 0.5 + 0.5 μL of 10 μM L15995 + H16498, 0.2 μL of 5 U/μL AmpliTag Gold DNA polymerase (Applied Biosystems, USA), 0.1–1 ng of DNA, and H₂O to a final volume of 25 μL. The PCR program was as follows: 95 °C for 10 min, 40× (95 °C for 15 s, 55 °C for 30 s, 72 °C for 1 min), and 72 °C for 30 min.
PCR was performed using a MasterCycler Nexus gradient thermocycler (Eppendorf, Germany). The resulting amplicons were visualized using agarose gel electrophoresis. DNA clean-up of the amplified fragments excised from the gel was performed using a Zymoclean Gel DNA recovery Kit (ZymoResearch, USA). Sanger sequencing was performed using a SeqStudio 3200 Genetic Analyzer (Applied Biosystems, USA). Raw data processing was performed using Sequencing Analysis Software v6.0 (Applied Biosystems, USA).