RNA was extracted using RNeasy Plus Mini kit (Qiagen) as per manufacturer’s instructions. TP53 complementary DNA was generated by RT-PCR using Superscript III One Step RT-PCR Platinum Taq HiFi (Thermofisher). PCR products were cloned into a pBluescript SK-vector and transformed into XL1-Blue competent cells. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen) and sequenced using the following primers (5′-CAC CAG CAG CTC CTA CAC CG-3′, 5′-ATG AGC GCT GCT CAG ATA GCG-3′, 5′-CGG CTC ATA GGG CAC CAC C-3′, 5′- TCT TCT TTG GCT GGG GAG AGG-3′). Tumour sequences were aligned using Seqman Pro (DNASTAR).
Superscript 3 one step rt pcr platinum taq hifi
Manufactured by Thermo Fisher Scientific
SuperScript III One-Step RT-PCR Platinum Taq HiFi is a laboratory equipment product for performing reverse transcription and polymerase chain reaction in a single-step process. It combines the SuperScript III reverse transcriptase and Platinum Taq DNA polymerase HiFi for efficient and high-fidelity amplification of RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy plus mini kit, by Qiagen (3 mentions)
Qiaprep spin miniprep kit, by Qiagen (3 mentions)
High pure viral rna kit, by Roche (3 mentions)
Seqman pro, by DNASTAR (2 mentions)
3 full race core set ver 2, by Takara Bio (2 mentions)
5 full race kit, by Takara Bio (2 mentions)
Abi prism 310 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Qiamp viral rna mini kit, by Qiagen (1 mentions)
Bioanalyzer dna 1000 chip, by Agilent Technologies (1 mentions)
S2 focused ultrasonicator, by Covaris (1 mentions)
Microtube, by Covaris (1 mentions)
Hygromycin b, by Merck Group (1 mentions)
9 protocols using superscript 3 one step rt pcr platinum taq hifi
1
TP53 Gene Sequencing Protocol
2
TP53 cDNA Sequencing from Tumor Samples
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RNA was extracted using RNeasy Plus Mini kit (Qiagen) and TP53 cDNA generated by RT-PCR using Superscript III One Step RT-PCR Platinum Taq HiFi (Thermofisher). PCR products were cloned into a pBluescript SK- vector and transformed into XL1-Blue competent cells. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen) and sequenced using the following primers (5′-CAC CAG CAG CTC CTA CAC CG-3', 5'-ATG AGC GCT GCT CAG ATA GCG-3′, 5′-CGG CTC ATA GGG CAC CAC C-3′, 5′-TCT TCT TTG GCT GGG GAG AGG-3′). Tumour and stromal sequences were aligned using Seqman Pro (DNASTAR).
3
Determining PRRSV Strain Identity in Tonsils
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For determining whether the virus isolated from tonsils was the immunization or the challenge PRRSV isolate, ORF5 from positive samples was amplified and sequenced. For that purpose, total RNA was obtained using QIAmp® Viral RNA Mini kit (Qiagen) following the manufacturer’s instructions. Then, PRRSV ORF5 was amplified by RT-PCR using a commercial kit (SuperScript III OneStep RT-PCR platinum TaqHiFi®, Invitrogen), following the manufacturer’s instructions. For this purpose, two different pairs of primers were used, one to amplify all PRRSV-1 viruses and another one to amplify the PRRSV-2 isolate used in the study (31 (link), 32 (link)). RT-PCR products were purified using a commercial kit (QIAQuick® Purification Gel Kit, Qiagen) following the manufacturer’s instructions. Individual sequences of both strands of the PCR products were determined with the same pair of primers used for RT-PCR, amplifying the samples by asymmetric PCR with fluorescent terminators and analyzing the products by electrophoresis on an ABI prism 310 Genetic Analyzer (Applied Biosystems). Sequences were manually corrected, purged of errors, and a bioinformatics analysis was performed. Sequences were aligned using Clustal Omega software (33 (link)).
4
Amplification of IBV S1 Gene
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The primer sets (Oligo5 [forward]: 5’-TGA AAA CTG AAC AAA AGA CA-3’ and CK2 [reverse]: 5’-CTC GAA TTC CNG TRT TRT AYT GRC A-3’) were designed for the IBV S1 gene, which contains the hypervariable region 1 and most of the hypervariable region 2 [17 (link)]. The S1 gene was amplified with the Superscript® III One-Step RT-PCR Platinum®Taq HiFi (Invitrogen™ Life Technologies™, Carlsbad, CA), which generated a PCR product of 344 bp. For the PCR reaction, the following reaction mix was made: 35 µL Buffer 10×, 14 µL dNTP, 14 µL CK2, 14 µL Oligo5, 243.25 µL H2O, 1.75 µL Taq polymerase (Taq HIFI, Invitrogen™ Life Technologies™, Carlsbad, CA), and 2 µL cDNA to a final volume of 25 µL. The PCR parameters were as follows: 45 cycles of denaturation (94°C, 20 s), annealing (51°C, 1 min), and polymerization (72°C, 1 min). The pre-denaturation step was done at 94°C for 5 min and post-polymerization step at 70°C for 10 min. A 20 µL aliquot of each PCR product was electrophoresed at 120 V for 40 min in 1.5% agarose gel, which was then stained with GelRed™ (Genaxxon Bioscience, Ulm, Germany, GmbH).
5
Amplicon-seq for HCV Genome Profiling
6
Viral Genome Sequencing Protocol
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Viral genomic RNA was extracted using High Pure Viral RNA Kit (Roche Applied Science) and amplified by RT-PCR with SuperScript III One-Step RT-PCR Platinum Taq HiFi (Invitrogen). Ten pairs of overlapping specific RT-PCR primers were designed based on sequences of PPMV-1 strains which were available in GenBank. For the 3’ leader and 5’trailer, primers were designed based on specific sequences of the six viruses, and the RNA was amplified using 3’-Full RACE Core Set Ver.2.0 (Takara) and 5’-Full RACE Kit (Takara) respectively according to the manufacturer’s instructions. Primer sequences used to amplify genome and 3’, 5’ ends are shown in S1 and S2 Tables. The amplified products were sequenced at Beijing Genomics Institute, China.
7
Reconstituting p38α Function in MAPK14-/- Cells
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To reconstitute p38α function in MAPK14−/−, a p38α cDNA (Accession#: NM_139012) was amplified by RT-PCR using Superscript III One Step RT-PCR Platinum Taq HiFi (Invitrogen) with RNA template extracted from HCT116 cells using RNeasy Plus Mini kit (QIAGEN). This PCR product was cloned into pcDNA5/FRT/TO (Invitrogen) and transformed into XL1-Blue competent cells. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (QIAGEN) and co-transfected with pOG44 into GFP-p53 MAPK14−/− HCT116 Flp-In T-REx™ cells. Following selection in 400 μg/ml hygromycin B (Sigma), colonies were pooled and expanded to create an isogenic polyclonal cell line. Expression of p38α by the addition of tetracycline hydrochloride (1 μg/ml) was confirmed by immunoblotting.
8
Comprehensive NDV Genome Sequencing Protocol
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The RNA extraction and RT-PCR were conducted as described previously [13 (link)]. Viral genomic RNA was extracted using High Pure Viral RNA Kit (Roche Applied Science) and amplified by RT-PCR with SuperScript III One-Step RT-PCR Platinum Taq HiFi (Invitrogen). Ten pairs of overlapping specific RT-PCR primers were designed based on the available NDV sequences from GenBank. For the 3’ leader and 5’trailer, primers were designed based on the specific sequence of duck/Guangxi/1261/2015, and the RNA was amplified using 3’-Full RACE Core Set Ver.2.0 (Takara) and 5’-Full RACE Kit (Takara) respectively according to the manufacturer’s instructions. Primer sequences used to amplify the genome are shown in S1 and S2 Tables. The amplified products were sequenced at Beijing Genomics Institute, China.
9
Viral RNA Extraction and Sequencing of NDV
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Viral genomic RNA was extracted using High Pure Viral RNA Kit (Roche Applied Science, Indianapolis, USA). The F and HN genes of six class I NDVs isolated from different provinces were amplified by RT-PCR with SuperScript III One-Step RT-PCR Platinum Taq HiFi (Invitrogen) with primers (pairs 3–6) that have been described previously [13 (link)]. The amplified products were sequenced at Beijing Genomics Institute, Beijing, China.
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