A 5’RACE was performed using the 5’ RACE System for Rapid Amplification of cDNA Ends, version 2.0 kit (Invitrogen, USA). Briefly, cDNA was generated by reverse transcription from total RNA extract followed by degradation of the RNA. dC-tailing was then performed with the cDNA and the resulting dC-tailed DNA was used as the template in PCR as described in the kit instructions. The PCR products were analyzed by agarose gel electrophoresis (1% agarose in TAE buffer). To identify the transcription start sites, PCR products were inserted into the pGEM-T easy vector kit as described by the manufacturer (Promega, USA). Insert were then PCR-amplified and the resulting PCR products were sequenced.
5 race system for rapid amplification of cdna ends version 2.0 kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 5' RACE System for Rapid Amplification of cDNA Ends, version 2.0 kit is a laboratory tool designed for the amplification of the 5' end of messenger RNA (mRNA) transcripts. The kit includes components necessary for reverse transcription, purification, and subsequent PCR amplification of the 5' end of cDNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Pgem t easy vector kit, by Promega (1 mentions)
Smarter race cdna amplification kit, by Takara Bio (1 mentions)
Pgem t, by Promega (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
S n a p column, by Thermo Fisher Scientific (1 mentions)
Pcr4 vector, by Thermo Fisher Scientific (1 mentions)
12 protocols using 5 race system for rapid amplification of cdna ends version 2.0 kit
1
Rapid Amplification of cDNA Ends
2
5' RACE Protocol for KRAS Gene
3
Determining Norovirus Genome 5' and 3' Ends
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To determine the 5′-ends of norovirus genomic RNA, rapid amplification of cDNA ends (RACE) was performed using the 5′ RACE System for Rapid Amplification of cDNA Ends Version 2.0 Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. Three primers (GSP1, GSP2 and nested GSP) were designed based on ORF1 of CMC-1 for RACE (Table 2 ). To obtain the exact sequence of the 3′-end of the genomic RNA, cDNA was synthesised by reverse transcription using 3′-oligo (dT)-anchor-R (Table 2 ). The second PCR was conducted using the ORF3_2F and 3′-anchor-R primers (Table 2 ) under the following conditions: 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, followed by 72 °C for 7 min.
4
Transcriptional Start Site Mapping of pecT mRNA
5
KRAS Gene 5'-RACE Amplification
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5′-RACE was performed using the 5′ RACE System for Rapid Amplification of cDNA Ends, version 2.0 kit (Invitrogen, Life Technology) with the manufacturer's instructions. Briefly, first-strand cDNA was synthesized using a kras -specific primer (5′-TCACATTTATTTCCTACTAGGACCATA-3′). The original mRNA template was removed by treatment with RNase Mix. Unincorporated dNTPs, GSP1, and proteins were separated from cDNA using a S.N.A.P. Column. A homopolymeric tail was then added to the 3′-end of the cDNA using TdT and dCTP. PCR was amplified by GSP2 primer (5′-GTACATCTTCAGAGTCCTTAACTCTT-3′) and deoxyinosine-containing anchor primer with cycling as described in the kit manual: 1 cycle of 94 °C for 4 min, then 35 cycles of 94 °C for 30 s, 55 °C for 30 s 72 °C for 1 min, and final extension of 72 °C for 10 min. From this reaction, 10 µl was analyzed on a 1.5% agarose gel.
6
Cloning of Carp TRIM32 Gene by RT-PCR and RACE
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To clone the full-length coding DNA sequence (CDS) of common carp trim32, RT-PCR and RACE techniques were used. Two pairs of degenerate primers corresponding to the highly conserved trim32 sequences from zebrafish (Danio rerio), Tilapia (Oreochromis spp.), Swordfish (Xiphias gladius), and Latimeria chalumnae were designed to clone the core section of the trim32 gene. Subsequently, the CDS region sequences were cloned through the RACE technique. 5’ RACE System for Rapid Amplification of cDNA Ends Version 2.0 Kit (Invitrogen, Shanghai, China) and SMARTer™ RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) were used to obtain the entire CDS region sequence of carp trim32. Next, specific primers were designed according to the sequence used to clone the full-length trim32 gene, the gene accession number was KX388359.
7
miRNA-directed cleavage site identification
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To confirm and identify miRNA-directed cleavage sites on target genes, total RNA isolations from root and bud in A. donax were performed as formerly described34 (link). 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 kit (Invitrogen) was used for 5′ race experiments according to manufacturer’s instructions. The PCR products amplified using second nested primers were gel purified, cloned into pGEM-T (Promega) and sequenced. All primers used are listed in Supplementary Table 1 .
8
Transcription Start Site Mapping
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Transcription start site mapping was conducted using a 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 Kit (Invitrogen, Life Technologies, USA) as described in the manufacturer’s guidelines. For this, RNA was isolated from cells under nitrogen starvation (N−) as described above, sequences for primers used can be found in Supplementary Table 4 .
9
5' RACE for Transcription Start Site Mapping
10
Determining SFTSV RNA Termini by RACE
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To determine the 5 -ends of the SFTSV genomic RNA, rapid amplification of cDNA ends (RACE) was performed using the 5 RACE System for Rapid Amplification of cDNA Ends Version 2.0 Kit (Invitrogen) according to the manufacturer's recommendations. Three primers, GSP1, GSP2, and nested GSP, were designed for 5 RACE PCR based on the L sequence (Table 1). To obtain the exact sequence of the 3 -end of the SFTSV genomic RNA, cDNA was synthesized by reverse transcription using 3 -oligo (dT)-anchor-R (Table 1). The second PCR was conducted using the S-F and 3 -anchor-R primers (Table 1) under the following conditions: 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min followed by 72°C for 10 min.
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