The TSS for tig was identified using the 5′ RACE System for Rapid Amplification of cDNA Ends, version 2.0 (Thermo Fisher Scientific). Primers are listed in Supporting Information Table S1 . In brief, first-strand cDNA synthesis was accomplished using 2 μg of B. burgdorferi total RNA and tig-specific primer 970R. cDNA purification and terminal deoxynucleotidyl transferase tailing were performed according to the manufacturer’s instructions. Tailed cDNA was then amplified by PCR using Taq polymerase (Thermo Fisher Scientific) with the Abridged Anchor Primer and tig-specific primer 971R, followed by a nested PCR amplification using the Abridged Universal Amplification Primer and tig-specific primer 916R. PCR product was then cloned into pCR2.1-TOPO (Thermo Fisher Scientific) and transformed into E. coli TOP10. Plasmid DNA was isolated using GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) and analyzed by DNA sequencing.
5 race system for rapid amplification of cdna ends version 2
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The 5' RACE System for Rapid Amplification of cDNA Ends, Version 2.0 is a laboratory equipment product designed to facilitate the rapid amplification of the 5' end of RNA transcripts. The system provides a set of reagents and protocols to efficiently generate full-length cDNA from known sequences.
Automatically generated - may contain errors
Lab products found in correlation
3 race system for rapid amplification of cdna ends, by Thermo Fisher Scientific (2 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Pcr2.1 topo, by Thermo Fisher Scientific (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Primescript rt reagent kit perfect real time, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Pureyield plasmid miniprep system, by Promega (1 mentions)
Platinum taq polymerase, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Genelute gel extraction kit, by Merck Group (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Poly a polymerase tailing kit, by Illumina (1 mentions)
Superscript reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Pgem t vector system, by Promega (1 mentions)
Primer premier 5, by Premier Biosoft (1 mentions)
Rna la pcrtm kit amv ver 1, by Takara Bio (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
23 protocols using 5 race system for rapid amplification of cdna ends version 2
1
5' RACE Analysis of Borrelia tig
2
5' RACE Protocol for Rapid Amplification of cDNA Ends
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Here 5′-RACE was performed using the 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions, with slight modifications. Briefly, 5 µg of human placental total RNA (BioChain Institute Inc., Newark, CA, USA) was reverse transcribed (RTed) using the gene-specific primer (GSP) 1 (primer i in Figure 1 a), and the RT products were column purified after RNase treatment. These purified single-stranded DNAs were used as a template for first polymerase chain reaction (PCR) after adding dA-tail using terminal nucleotidyl transferase (TdT). The second PCR product was gel-purified and sequenced by the Sanger method. The dT adaptor used in the first PCR and the adaptor primer used in the second PCR are shown in Table 1 , along with the GSPs (primers i and j).
3
Rapid 5' RACE Amplification
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5′RACE was conducted using 5′RACE System for Rapid Amplification of cDNA Ends, version 2.0 (Life Technologies). Primers used in PCR are listed in Supplemental Table S2. PCR products were resolved by 2% agarose gel electrophoresis and DNA fragments purified using QIAquick Gel Extraction Kit (Qiagen) and cloned into pGEM-T Easy Vector (Promega). Four colonies from each of the isoforms were picked and sequenced.
4
Rapid 5'UTR Sequencing of HCV
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For a rapid identification of 5’UTR sequence of HCV, RNAs extracted from 100 μl of virus-containing supernatants or PBMCs were amplified by using a PrimeScript RT reagent Kit (Perfect Real Time) (Takara Bio) and 5’RACE was performed by using a 5'RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Life Technologies) as described by Ono et al. [23 (link)].
5
Rapid Identification of HCV 5'UTR
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For a rapid identification of 5’UTR sequence of HCV, RNA was extracted from 100 μl of virus-containing supernatants or PBMCs. The first-stranded cDNA was synthesized by using a PrimeScript RT reagent Kit (Perfect Real Time) (Takara Bio) and the 5’UTR of HCV was amplified. 5’RACE was performed by using a 5'RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Life Technologies) as described by Li et al. [44 (link)] with modification (see S2 Table ).
6
Rapid Amplification of cDNA Ends (RACE) Protocol
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3’-RACE was performed using the 3’-RACE System for Rapid Amplification of cDNA ends (Life Technologies), according to the manufacturer’s protocol, in 20 μl reactions containing 200 ng of total RNA template. 5’-RACE was performed using the 5’-RACE System for Rapid Amplification of cDNA ends, Version 2.0 (Life Technologies), according to the manufacturer’s protocol, in 25 μl reactions containing 500 ng of total RNA template and 2.5 pmol of gene-specific primer. Primary and nested PCR reactions were performed using Platinum Taq polymerase (Life Technologies), according to the manufacturer’s protocol, in 50 μl reactions containing 500 nM gene-specific primer. A list of primers used in the study is provided in S1 Fig . PCR products were cloned using the TOPO TA Cloning Kit (Life Technologies) according to the manufacturer’s protocol. Individual colonies were grown in L-Broth, and plasmids were purified using the PureYield Plasmid Miniprep System (Promega) and then sequenced (Source BioScience).
7
Characterizing Synthetic Promoter Variants
8
Rapid Amplification of miRNA Targets
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To obtain cleavage fragments resulting from transcript processing by microRNAs, we used the 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen). Total RNA was extracted from 10-day-old 35S::OR seedlings using the TRIZOL method described above. The oligo(dT) was then used for cDNA synthesis. Initial PCR was carried out using the 5′ RACE Abridged Primer and gene specific outer primer (5′-GTA ATG CAA TTG TAC TCT CGA G-3′). Nested PCR was carried out using 1/100 of the initial PCR reaction as template, Universal Amplification Primer, and gene-specific inner primer (5′-AGA GAA TCG GAA GTT AAC AAA ATA G-3′). RACE fragments were cloned and sequenced after gel purification.
9
5' RACE Analysis of Mosquito Transcripts
10
Transcriptome analysis of E. coli O157:H7 under salt stress
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An overnight culture of Escherichia coli O157:H7 strain Sakai (GenBank accession NC_002695, EHEC) [22 (link)] was inoculated 1:100 in 0.5 × LB with 400 mM NaCl and incubated at 37 °C and 150 rpm until an OD600 of 0.8 was reached. Total RNA of 500 μl EHEC culture was isolated with Trizol and the remaining DNA was digested using 2 U TURBO™ DNase (Thermo Fisher Scientific). The 5’RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen) was used according to the manual. After the second polymerase chain reaction (PCR), the dominant product was excised from the agarose gel and purified with the GenElute™ Gel Extraction Kit (Sigma-Aldrich). The PCR product was Sanger sequenced by Eurofins with oligonucleotide laoB+25R.
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