Total RNA was extracted from wild-type fly testes using Trizol reagent (Sangon), then the amount of 1 µg was incubated with PrimeScript RTase(50 U/µl) to transcribe cDNA, which was used as the template in PCR reactions, according to the manufactures’ protocol (PrimeScript High Fidelity RT-PCR Kit, Takara). The PCR primers were designed to explore the different gish mRNA splicing variants (S1 Table). Total RNAs of fly testes were independently isolated from each phenotype (wild-type and gish mutant) using the Trizol reagent (Sangon) and reverse transcribed into cDNA according to the manufactures’ protocol (PrimeScript RT reagent Kit with gDNA Eraser, Takara). For each independent cDNA sample, quantitative PCR was run on CFX96 Touch (BioRad) to measure total gish mRNAs with rp49 as reference according to the manufactures’ protocol (SYBR Premix EX TaqTM II qPCR Kit, Takara). The following primers were used in this study: gish, 5′-GCCGGTGGTAAAAGCTCAAG-3′ (sense) and 5′-CGCCAAAATTACCACAGCCA-3′ (antisense); rp49, 5′-CACTTCATCCGCCACCAGTC-3′ (sense) and 5′-CGCTTGTTCG ATCCGTAACC-3′ (antisense).
Primescript high fidelity rt pcr kit
Manufactured by Takara Bio
Sourced in Japan, United States
The PrimeScript High Fidelity RT-PCR Kit is a reagent kit designed for reverse transcription and subsequent high-fidelity polymerase chain reaction (PCR) amplification. The kit includes a reverse transcriptase enzyme and a high-fidelity DNA polymerase for sensitive and accurate RNA-to-cDNA conversion and amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Pcdh cmv mcs ef1 copgfp vector, by System Biosciences (3 mentions)
Lightcycler nano instrument, by Roche (3 mentions)
Qiaamp viral rna mini kit, by Qiagen (2 mentions)
Ion torrent pgm, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Ppackh1 packaging plasmid mix, by System Biosciences (2 mentions)
Faststart essential dna green master, by Roche (2 mentions)
Sybr premix ex taqtm 2 tli rnaseh plus kit, by Takara Bio (2 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Cfx96 touch, by Bio-Rad (1 mentions)
Trizol reagent, by Sangon (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
5 and 3 race kit, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Tri reagent, by Cosmo Bio (1 mentions)
34 protocols using primescript high fidelity rt pcr kit
1
Transcriptomic Analysis of gish Splicing
2
Genome Sequencing of Japanese PDCoV Strains
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Complementary DNA (cDNA) was synthesized from 7 out of 72 samples positive for PDCoV-specific PCR using the PrimeScript High Fidelity RT-PCR Kit (Takara Bio, Inc., Shiga, Japan) with a random 6-mer primer. Nearly complete genomes, consisting of eight overlapping amplicons (approximately 4 kb in length), were generated from the cDNAs using a set of primers originally designed with reference to PDCoV strains reported previously (Table S1). Eight amplicons were pooled in equal amounts, and analysed using next-generation sequencing technology (Ion Torrent PGM; Life Technologies, Carlsbad, CA, USA). The consensus genome sequences of the seven Japanese PDCoV strains were determined with reference to the complete genomes of the 17 U.S., two Hong Kong, and one Korea PDCoV strains assigned previously in GenBank. Phylogenetic analysis based on the complete genomes was performed using the maximum-likelihood method with the general time reversible nucleotide substitution model and 1000 bootstrap replicates implemented in the MEGA6 program (Tamura et al., 2013 (link)).
3
Cloning of BmABCA2 cDNA in pcDNA3.1-EGFP-Ble
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Total RNA was isolated from midgut tissue of 5th instar larvae of the wild-type silkworm strain, using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) according to the protocols of manufacturer, and used for cDNA synthesis as a template. The BmABCA2 cDNA was amplified by one-step RT-PCR using PrimeScript High Fidelity RT-PCR Kit (TaKaRa Bio). The amplified cDNA using primers (forward: 5′-CCACCCGGATCCGATATGAGACCTCAGAGAAAAGAAGCC-3′, reverse: 5′- GTCTTTGTAGTCGATCAAGCCTTCCCTTTGATATTTCGT-3′) was cloned into EcoRV site of the pcDNA3.1 (Thermo Fisher Scientific, Waltham, MA, USA) using In-Fusion® HD Cloning Kit (TaKaRa Bio, Kusatsu, Japan). The neomycin resistance gene of pcDNA3.1 was replaced by Enhanced green fluorescent protein (EGFP) - Streptoalloteichus hindustanus ble (Sh ble) fusion gene by In-fusion cloning method described below. The linearized vector was generated from pcDNA3.1 plasmid as a template by PCR using primers (forward: 5′-GCCCTTGCTCACCATGCGAACGATCCTCATCCTGTC-3′, reverse: 5′- GAGGAGCAGGACTGAGCGGGACTCTGGGGTTCG-3′). The EGFP-ble fusion gene was amplified using primers (forward: 5′-ATGGTGAGCAAGGGCGAGGAG-3′, reverse: 5′- TCAGTCCTGCTCCTCGGCCAC-3′) and cloned into the linearized vector.
4
Total RNA Isolation and RT-PCR
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The total RNA from each sample was isolated with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. RT-PCR was carried out with a PrimeScript High Fidelity RT-PCR kit (Takara, Japan) and the following primers:
5
PEDV Genome Sequencing and Phylogenetic Analysis
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Viral RNA was extracted from a 250 μL sample using the TRIzol Reagent (Takara, Shiga, Japan). The PEDV cDNA was synthesized by means of the PrimeScript High Fidelity RT-PCR Kit (Takara). Full-length PEDV genome amplification was performed with primers described previously [14 (link)]. The 5′ and 3′ end sequences were determined with the 5′ and 3′ RACE kit (Takara). For each amplicon, more than three independent clones were sequenced to determine the consensus sequence of a given genomic region.
The ClustalX (ver.1.81) software was used to align the full-length genome sequences. Phylogenetic analyses based on the S and open reading frame 3 (ORF3) genes or the entire genome were performed by the maximum-likelihood method with the general time-reversible nucleotide substitution model, where 1000 bootstrap replicates were implemented in the MEGA6.0 software [15 ].
The ClustalX (ver.1.81) software was used to align the full-length genome sequences. Phylogenetic analyses based on the S and open reading frame 3 (ORF3) genes or the entire genome were performed by the maximum-likelihood method with the general time-reversible nucleotide substitution model, where 1000 bootstrap replicates were implemented in the MEGA6.0 software [15 ].
6
Extraction and Isolation of C. depressa Genes
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Standard techniques were used for DNA manipulation [41 ]. Genomic DNA and total RNA were obtained from the pericarp of C. depressa fruits. Flesh pericarp was ground using a mortar and pestle in the presence of liquid nitrogen. Genomic DNA was extracted by the cetyltrimethylammonium bromide (CTAB) extraction method [42 (link)]. Total RNA was prepared using a TRI reagent (Cosmo Bio Co. Ltd., Tokyo, Japan) according to the manufacturer’s protocol. First-strand cDNA was synthesized using a PrimeScript High Fidelity RT-PCR Kit (TaKaRa, Shiga, Japan) with an oligo dT primer, and the products were used as PCR templates to isolate CdFOMT-coding genes.
7
Purification and Cloning of Schistosoma Antigens
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S. mansoni adult worms were stored in RNAlater (QIAGEN) on collection. The samples were then crushed using Tissue-Ruptor (QIAGEN). Total RNA was isolated from crushed adult worms using TRIzol plus RNA Purification kit (Ambion), according to the manufacturer’s instructions. The cDNAs of target regions were amplified by using PrimeScript High Fidelity RT-PCR Kit (Takara), according to the manufacturer’s instructions. Briefly, the first strand cDNA was synthesized from RNA with reverse transcriptase using oligo dT primer, and PCR was performed by using the reverse transcription reaction mixture as the template with a pair of specific primers for each candidate antigen. PCR products were purified using QIAEX II Gel Extraction Kit (QIAGEN). Sh-SERPIN cDNA was chemically synthesized by Integrated DNA Technologies, Inc. Introduction of G444A mutation in Sm-SERPIN was performed by overlap extension PCR. The cDNAs were then sub-cloned into pET-52b(+) vector (Novagen). Structure of the antigens and the fusion tags are summarized inTable 1 .
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S. mansoni adult worms were stored in RNAlater (QIAGEN) on collection. The samples were then crushed using Tissue-Ruptor (QIAGEN). Total RNA was isolated from crushed adult worms using TRIzol plus RNA Purification kit (Ambion), according to the manufacturer’s instructions. The cDNAs of target regions were amplified by using PrimeScript High Fidelity RT-PCR Kit (Takara), according to the manufacturer’s instructions. Briefly, the first strand cDNA was synthesized from RNA with reverse transcriptase using oligo dT primer, and PCR was performed by using the reverse transcription reaction mixture as the template with a pair of specific primers for each candidate antigen. PCR products were purified using QIAEX II Gel Extraction Kit (QIAGEN). Sh-SERPIN cDNA was chemically synthesized by Integrated DNA Technologies, Inc. Introduction of G444A mutation in Sm-SERPIN was performed by overlap extension PCR. The cDNAs were then sub-cloned into pET-52b(+) vector (Novagen). Structure of the antigens and the fusion tags are summarized in
8
RNA Extraction and RT-qPCR Analysis
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Total RNA of FACS-purified HCT116 transfectants was prepared using RNeasy Mini kits (Qiagen). RT-qPCR analysis was performed using PrimeScript High Fidelity RT-PCR kit (Takara Bio Inc) with SYBYR Gold (Thermo Fisher Scientific) and a CFX96 Real-Time system instrument (Bio-Rad, Hercules, CA, USA).
9
Rhodosporidium toruloides RNA Extraction
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Example 1
The cell pellets harvested from 1 ml culture broth of Rhodosporidium toruloides in YPD medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose) were immediately frozen in liquid nitrogen and stored at −70° C. Total RNA was isolated from about 30-50 mg cell samples using the FastRNA Pro Red Kit and FastPrep Instrument (Qbiogen, Inc., Irvine, USA) following the manufacturer's instructions, and the setting of FastPrep Instrument was 6.0 m/s for 60 s. The RNA concentration and quality were determined by Nanodrop ND1000 Spectrophotometer (ThermoFisher Scientific), while the RNA integrity was assessed by agarose gel electrophoresis. cDNA was synthesized by PrimeScript™ High Fidelity RT-PCR Kit (Takara Bio Inc.). Genomic DNA of Saccharomyces cerevisiae and Aplanochytrium kerguelense were extracted as described before (Burke, D., Dawson, D. & Stearns, T. (2000) Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual, Cold Spring Harbor Laboratory Press, Plainview, N.Y.). Gene specific primers (Table 3) were used to amplify the open reading frame (ORF) of FAS genes.
10
Lentiviral Transduction of IDH2 and PLCB1 Mutants
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Total RNA was extracted and purified from IDH2 WT and IDH2 mut TF‐1 cells with the RNeasy Micro Kit (Qiagen). RNA was reverse transcribed and mutant form of IDH2 and PLCB1 genes were amplified using the PrimeScript High Fidelity RT‐PCR Kit (Takara Bio Inc.). The PCR product was gel‐purified and cloned into pENTR/D‐TOPO vector using the pENTR/D‐TOPO Cloning Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The target gene sequences were further cloned into pMAL (a gift from John Dick and Peter van Galen [Addgene plasmid #161783; http://n2t.net/addgene:161783 ; RRID:Addgene_161,783])
25 (link) using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific). The generated lentiviral vector plasmids were cotransfected with lentiviral packaging vectors into HEK293 T cells using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) and viral supernatants were harvested and concentrated by Lenti‐X Concentrator (Takara Bio Inc.). The lentiviral supernatant was added to IDH2 mut TF‐1 or THP‐1 cell cultures in the presence of 8 μg/mL protamine sulfate and centrifuged at 800 g for 1 h at room temperature, followed by incubation at 37°C for 16 h. The transduced cells were expanded and GFP‐positive cells were used for subsequent experiments. Empty lentiviral vector‐transduced cells were used as control.
25 (link) using Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific). The generated lentiviral vector plasmids were cotransfected with lentiviral packaging vectors into HEK293 T cells using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) and viral supernatants were harvested and concentrated by Lenti‐X Concentrator (Takara Bio Inc.). The lentiviral supernatant was added to IDH2 mut TF‐1 or THP‐1 cell cultures in the presence of 8 μg/mL protamine sulfate and centrifuged at 800 g for 1 h at room temperature, followed by incubation at 37°C for 16 h. The transduced cells were expanded and GFP‐positive cells were used for subsequent experiments. Empty lentiviral vector‐transduced cells were used as control.
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