The 5'-RACE was carried out with a 5'full RACE kit (TaKaRa Code: D315) following manufacturer instructions. Total RNA was extracted from the second flower. mRNA was H.P. Si et al. dephosphorylated and the mRNA cap structure was removed. A 5'-RACE adaptor was ligated to the decapped mRNA with a 5'-full RACE kit (TaKaRa) and the first-stranded cDNA was synthesized by reverse transcription. The first-round PCR protocol consisted of initial denaturation at 94°C for 4 min, followed by 32 cycles at 94°C for 45 s, 58.7°C for 45 s, and 72°C for 60 s, and a final extension of 7 min at 72°C. The second-round PCR with nested primers consisted of 4 min at 94°C, followed by 32 cycles at 94°C for 45 s, 59°C for 45 s, and 72°C for 60 s, and a final extension of 7 min at 72°C.
5 full race kit
Manufactured by Takara Bio
Sourced in Japan, China
The 5'-Full RACE Kit is a laboratory tool used for rapid amplification of cDNA ends (RACE) to obtain the full-length sequence of a target mRNA. It provides a simple and efficient method to obtain the 5' end of a transcript starting from a partial sequence. The kit includes reagents and protocols necessary for performing the 5' RACE procedure.
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Lab products found in correlation
Pmd18 t vector, by Takara Bio (16 mentions)
3 full race core set ver 2.0 kit, by Takara Bio (9 mentions)
3 full race kit, by Takara Bio (8 mentions)
Rnaiso plus reagent, by Takara Bio (5 mentions)
3 full race core set ver 2, by Takara Bio (4 mentions)
3 full race core set, by Takara Bio (4 mentions)
Abi 3130 genetic analyzer, by Thermo Fisher Scientific (3 mentions)
Qiaquick gel extraction kit, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Gel extraction kit, by Sangon (3 mentions)
Pmd19 t vector, by Takara Bio (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Pgem t vector, by Promega (2 mentions)
Peasy blunt cloning vector, by Transgene (2 mentions)
Pcr product recovery kit, by Takara Bio (2 mentions)
Dh5α competent cells, by Takara Bio (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
High pure viral rna kit, by Roche (2 mentions)
Superscript 3 one step rt pcr platinum taq hifi, by Thermo Fisher Scientific (2 mentions)
Biospin gel extraction kit, by BioFlux (2 mentions)
80 protocols using 5 full race kit
1
5'-RACE Transcription Analysis Protocol
2
Subcloning and Sequencing of VL and VH Domains
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The genes of V light-chain (VL) and V heavy-chain (VH) domains from each hybridoma were subcloned using 5'-Full RACE kits (Takara Bio, Kyoto, Japan). The amplified DNA was directionally subcloned into a plasmid vector using the TOPO TA cloning kit (Invitrogen) and sequenced using a 3130xl Genetic Analyzer (Applied Biosystems, Carlsbad, CA).
3
Rapid Amplification of cDNA Ends for RNA Characterization
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5′-rapid amplification of cDNA ends (RACEs) used 5′-Full RACE kits (Takara) according to the manufacturer’s instructions. Briefly, 5 μg of total RNA was treated with 10 units of calf intestine alkaline phosphatase (1 h, 50°C) and 1 unit of tobacco acid pyrophosphotase (1 h, 37°C) and ligated to 250 pmol of supplied 5′ RACE adaptor with 40 unit of T4 RNA ligase (1 h, 16°C). Reverse transcription was at 42°C for 1 h with 5 units M-MLV reverse transcriptase and 25 pmol antisense RNA specific primer (5′ RACE RT). The cDNA was amplified with an adaptor-specific primer (5′ RACE outer primer) and asrC specific primer (5′ RACE GSP1). A second amplification was with 5′ RACE inner primer and 5′ RACE GSP2 using first PCR products as template. Purified PCR products were cloned into pGEM-T vector (Promega). Bacterial colonies were checked for appropriate inserts by PCR and confirmed by sequencing. The protocol for 3′-RACE was as described previously (Argaman et al., 2001 (link)). Briefly, total RNA (15 μg) was dephosphorylated with calf intestine alkaline phosphatase (Takara). RNA was ligated to 5′-phosphorylated 3′-RACE adaptor. Reverse transcription was performed as described for 5′-RACE with 3′-RACE adaptor-specific primer. Nested PCR was performed with 3′-RACE adaptor primer and asrC-specific primers (3′-RACE GSP1 and 3′-RACE GSP2). Cloning and sequence analysis were as described above.
4
Maize miRNA 5'RACE Assay
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We conducted RLM-5′RACE according to the method of Leonardo Alves [32] (link). Pre-zma-miR169 overexpression vectors (except for zma-miR169d/e) and overexpression vectors for seven potential targets with 3′UTR target sites were pooled and co-transfected into maize protoplasts. Total RNA was extracted for 5′RACE assays performed using a 5′-Full RACE Kit (D315; Takara, Kyoto, Japan). Total RNA was linked with an RNA adaptor and reverse transcribed using Reverse Transcriptase M-MLV with random 9-mers to produce cDNA. Nested-PCR was then performed using outer and inner primers to obtain the 3′-end degradation sequence. Amplification products were cloned into a pEASY-Blunt Cloning Vector (Beijing TransGen Biotech) for sequencing. The primers used for nested-PCR are listed in Table S4 .
5
Cloning and Characterization of IbBT4 from Sweet Potato
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Total complete RNA was isolated from in vitro-grown Xushu55-2 plants and then reverse transcribed to generate cDNA (Zhou et al., 2019 (link)). Based on the expressed sequence tag (EST) selected from the transcriptome sequencing data of Xushu55-2 (Zhu et al., 2018 (link)), the 5′-untranslated region (UTR) and 3′-UTR of IbBT4 were amplified using rapid amplification of cDNA ends (RACE) procedure using 5′- Full RACE Kit and 3′-Full RACE Core Set Ver.2.0 Kit (TaKaRa, Beijing, China). The cDNA sequence was analyzed by NCBI1 . The coding sequence (CDS) was cloned by PCR with specific primers. Its genomic sequence was cloned from Xushu55-2 genomic DNA via PCR in conjunction with specific primers. The promoter region was cloned with Universal Genome Walker 2.0 Kit (TaKaRa, Dalian, China). All of the specific primers are listed in Supplementary Table S1 . IbBT4 was annotated in the NCBI databases2 . Amino acid sequence alignments, phylogenetic relationships and exon-intron structure were analyzed with DNAMAN software, MEGA 7.0 software and the Splign tool, respectively, and the molecular weight and theoretical isoelectric point (pI) were calculated via online software (Kang et al., 2019 (link)). The cis-acting regulatory elements in the promoter region were analyzed via the PlantCARE database.
6
Validation of Candidate Targets via RLM 5'-RACE
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For the validation of candidate targets, modified RNA Ligase-Mediated 5′-RACE (RLM 5′-RACE) was performed. 5′-Full RACE Kit (Takara) was used according to the manufacturer's instructions, with slight modifications. Briefly, total RNA was directly ligated to the 5′ adaptor followed by reverse transcription with the oligo (dT) primers. PCR was performed with 5′ primers and 3′ gene-specific primers using the cDNA as the template (Table S1 ). The 5′-RACE PCR products were purified using Takara PCR product recovery kit, cloned, and sequenced.
7
Whole Genome Sequencing of Viral Isolates
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The full-length genome sequences of the virus were amplified by the “primer-walking” strategy, which was used to close the gaps as necessary7 (link). Briefly, overlapping fragments representing whole genomes were amplified by RT-PCR using specific primers (Table S3 ). The RT-PCR products were purified for sequencing using the QIAquick Gel extraction kit (Qiagen, Hilden, Germany), and the amplicons were sequenced on an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) as described above. The 5′ end of the genome was amplified based on the manufacturer’s instructions with the 5′-Full RACE Kit (Takara, Shiga, Japan). The 3′ end of the genome was amplified using an oligo-dT primer (7500 A) previously reported in another study46 (link).
8
Cloning and Sequencing of AcGA2ox Gene
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First strand cDNA was synthesized using RevertAidTM First Strand cDNA Synthesis Kit according to the manufacturer’s protocol. cDNA was used as a template for amplifying a partial sequence of the AcGA2ox gene. The forward and reverse primers were 5′-GGA(G) TTC TTC(T) AAA(G) GTC (GT) A(G)TA(CGT) AAC(T) CA -3′ and 5′-TA(CT)A CA(G)C TCT T A(G)A AC(T)C TC(T)C CA(G)T T-3′, respectively. Conditions for PCR amplification: 35 cycles of 94 °C for 1 min, 45 °C for 1.5 min and 72 °C for 2 min.
The 5′- and 3′- ends of the AcGA2ox cDNA were obtained by RACE-PCR, using the 5′-Full RACE Kit and the 3′- RACE version2.0 (TaKaRa, Shiga, Japan). For the 5′-RACE, the primers for the 1st nested PCR and the 2nd nested PCR amplifications were: 5′-AGA GCT CCT TCT CGA CCT GTG GCA-3′ (2 × 2-5outer), 5′-ACC TCA ATG CTT CGT CCT CCA AC-3′ (2 × 2-5inner), respectively. Those for 3′-RACE were 5′-TCT GTT CCT CCT GAT CAA AGC TCT-3′ (2 × 2-3outer), 5′-TCT TCA TCA ATG TTG GCG ATT CA-3′ (2 × 2-3inner), respectively. The PCR products were separated in 1.0% (w/v) agarose gels, purified and ligated into the pMD-18T vector (TaKaRa, Shiga, Japan). The nucleotide sequences of the cDNA inserts were analyzed using the thermo sequenase dye terminator cycle sequencing kit and a 377 DNA sequencer (Perkin-Elmer Applied Biosystems).
The 5′- and 3′- ends of the AcGA2ox cDNA were obtained by RACE-PCR, using the 5′-Full RACE Kit and the 3′- RACE version2.0 (TaKaRa, Shiga, Japan). For the 5′-RACE, the primers for the 1st nested PCR and the 2nd nested PCR amplifications were: 5′-AGA GCT CCT TCT CGA CCT GTG GCA-3′ (2 × 2-5outer), 5′-ACC TCA ATG CTT CGT CCT CCA AC-3′ (2 × 2-5inner), respectively. Those for 3′-RACE were 5′-TCT GTT CCT CCT GAT CAA AGC TCT-3′ (2 × 2-3outer), 5′-TCT TCA TCA ATG TTG GCG ATT CA-3′ (2 × 2-3inner), respectively. The PCR products were separated in 1.0% (w/v) agarose gels, purified and ligated into the pMD-18T vector (TaKaRa, Shiga, Japan). The nucleotide sequences of the cDNA inserts were analyzed using the thermo sequenase dye terminator cycle sequencing kit and a 377 DNA sequencer (Perkin-Elmer Applied Biosystems).
9
Rapid Amplification of cDNA Ends
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RLM-5’ RACE was performed using 5’-Full RACE Kit (Takara, Japan). Briefly, 5 µg total RNA was ligated to RNA adapter followed by reverse transcription using the Oligo (dT) primer and superscript reverse transcriptase. PCR was performed with 3’ gene-specific primers and 5’ adaptor primers. The PCR product was cloned into pEASY-T1 vector and plasmid DNA was sequenced.
10
RNA Ligase-Mediated 5'-RACE for Spikelets
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To conduct the RNA ligase–mediated 5’-RACE, 1 μg total RNAs from equal mixture of superior and inferior spikelets at 10DAF, 15DAF, 21DAF, 27DAF, and 35DAF was ligated to a 5’-RACE RNA adapter without calf intestine alkaline phosphatase treatment using 5′-Full RACE Kit (Takara), followed by a reverse transcription reaction. Then 1 μl of reverse transcription product was used as template to amplify the 5’ end of the potential target by using the outer 5’ RNA adaptor primer supplied in the kit and outer 3’ gene-specific primer. Thirty more cycles of PCR were further carried out using the inner 5’ RNA adaptor primer supplied in the kit and inner 3’ gene-specific primer. The final PCR product was showed on the ethidium bromide-stained agarose gel, cloned into pGEM-T Easy vector, and at least 7 independent clones from each reaction were sequenced finally.
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