5 race system for rapid amplification of cdna ends

Manufactured by Thermo Fisher Scientific

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The 5' RACE System for Rapid Amplification of cDNA Ends is a laboratory tool designed to facilitate the amplification and identification of the 5' end of cDNA sequences. It provides a method for extending known sequences to their full-length 5' ends.

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5′ RACE system (50 citations) 5′ RACE (23 citations) 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (23 citations) 5′ RACE System for Rapid Amplification of cDNA Ends kit (15 citations) RACE system (12 citations) 5′ RACE System for Rapid Amplification of cDNA Ends, version 2.0 kit (12 citations) 5′ and 3′ RACE System for Rapid Amplification of cDNA Ends (5 citations) 5′ RACE system for rapid amplification of cDNA (3 citations)

32 protocols using 5 race system for rapid amplification of cdna ends

Ostrich Toll-like Receptor Cloning and Expression

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Total RNA was isolated from thymus using TRIzol reagent (TaKaRa). First strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) and DNase I (Thermo Scientific). Degenerative PCR primers for ostrich TLR4 and TLR3 were designed based on the multiple alignments of previously reported sequences in birds (S2 Table). The first PCR reactions were performed with the degenerative primers (Table 1) to obtain the partial sequences of ostrich TLR3 and TLR4. Next, nested primers for ostrich TLR4 (Table 1) were designed from the obtained sequence to perform 5’ and 3’RACE using 5’ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, USA) and Smart RACE cDNA amplification kit (Clontech, USA), respectively. The RACE products were ligated into pMD18-T vector, sequenced and assembled to obtain the full-length ostrich TLR4 cDNA.
The qRT-PCR primers for TLR3, TLR4 and actin were designed from our cloning results (KM651767, KM408431 and KJ729106.1), since the ostrich genome was not available when we started the research. The qRT-PCR primers for p100, IRF1, IL6 and IFN were designed from subsequent ostrich genomic information (KFV79432.1, KFV73539.1, KFV82123.1 and KFV87588.1).

Huang H.B., Xiao K., Lu S., Yang K.L., Ansari A.R., Khaliq H., Song H., Zhong J., Liu H.Z, & Peng K.M. (2015). Increased Thymic Cell Turnover under Boron Stress May Bypass TLR3/4 Pathway in African Ostrich. PLoS ONE, 10(6), e0129596.

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Cloning and Validation of Plasmid Constructs

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pLKO.1, pCDH, and pCMV were used in our study. ShRNA sequences were generated by PCR and then cloned into the pLKO.1 vector. The KRT80 and lncHNSCAT1 overexpression cassette was generated by PCR, cloned into the pCDH vector, and verified by DNA sequencing. A rapid amplification of cDNA ends (RACE) assay was carried out with the 5′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, #18374-058) and the 3′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, #18373-019) according to the manufacturer's protocols. The amplified DNA fragment was cloned into the pGEM-T Easy vector and validated by Sanger sequencing (Sangon Biotech, China).

Zhao Y., Huang X., Zhang Z., Li H, & Zan T. (2022). The Long Noncoding Transcript HNSCAT1 Activates KRT80 and Triggers Therapeutic Efficacy in Head and Neck Squamous Cell Carcinoma. Oxidative Medicine and Cellular Longevity, 2022, 4156966.

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Cloning and Sequencing of Goose DBH Gene

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Total RNA was extracted from collected tissue samples using TRIzol reagent according to the manufacturer’s instruction (TaKaRa, China) and re-suspended in RNase-free water. The concentration and purity were determined with a NanoDrop Spectrophotometer (NanoDrop, USA). After purification, 2 μg of total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega, USA) according to the manufacturer’s protocol. Primers were designed according the unigene (Xu et al., [18 (link)]; Additional file 1) and reverse transcription PCR (RT-PCR) was performed using ovarian cDNA from geese. The PCR product was purified, cloned into the pMD19-T vector (TaKaRa, China), and subjected to sequence analysis. The 5′- and 3′-ends of DBH were amplified via rapid amplification of cDNA ends (RACE) using the 5´-RACE System for Rapid Amplification of cDNA Ends (Invitrogen, USA) and the 3′-Full RACE Kit (TaKaRa, China), respectively. RACE primers (Additional file 1) were designed using the partial DBH nucleotide sequence obtained from RT-PCR. Touchdown and nested PCRs were performed according to the manufacturer’s instructions. Amplicons were then cloned into a plasmid vector for nucleotide sequencing by Sangon Biotech (Shanghai, China).

Xu Q., Song Y., Liu R., Chen Y., Zhang Y., Li Y., Zhao W., Chang G, & Chen G. (2016). The dopamine β-hydroxylase gene in Chinese goose (Anas cygnoides): cloning, characterization, and expression during the reproductive cycle. BMC Genetics, 17, 48.

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5' RACE for Transcriptomic Exploration

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5′ RACE experiments were performed with 5′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen) using three gene-specific primers (GSP) that anneal to the known region and an adapter primer that targets the 5′ end. Products generated by 5′ RACE were subcloned into TOPO-TA vectors and individual colonies were sequenced.

Fiszbein A., Krick K.S., Begg B.E, & Burge C.B. (2019). Exon-mediated activation of transcription starts. Cell, 179(7), 1551-1565.e17.

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5'RACE Analysis of Gene Expression

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The 5′-RACE analysis was performed using the 5′RACE System for Rapid Amplification of cDNA Ends (Invitrogen) according to the manufacturer’s recommendations. The oligonucleotides used for the 5′-RACE analysis are listed in Table S1. DNase I-treated total RNA was reverse-transcribed into cDNA using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific) at 60 °C for 1 hour. The obtained cDNA was lengthened by terminal deoxynucleotidyl transferase (Thermo Scientific), followed by PCR amplification with PCR Master Mix (2X) (Thermo Scientific). The following program was used: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 10 min. The obtained amplicons were loaded on a 1.5%-agarose gel, and the products were excised for purification (Zymoclean Gel DNA Recovery Kit), which was followed by sequencing.

Rubtsova M.P., Vasilkova D.P., Moshareva M.A., Malyavko A.N., Meerson M.B., Zatsepin T.S., Naraykina Y.V., Beletsky A.V., Ravin N.V, & Dontsova O.A. (2019). Integrator is a key component of human telomerase RNA biogenesis. Scientific Reports, 9, 1701.

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Determining Transcription Start Site of SGIV ICP46

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Transcription start site (TSS) of SGIV ICP46 mRNA was determined using 5′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First strand cDNA was synthesized from 3 μg total RNA isolated at 12 h p.i. using a specific reverse primer (GSP1, 5′-CAA AGG GTG TGG CAA G-3′). After RNAase treatment and cDNA purification, an oligo (dC) tail was added at the 5′ end. The resulting product was PCR amplified with a universal primer containing a poly-dG sequence and specific oligonucleotides (GSP2, 5′-TTT CCG CAG TGT ACG AGT CC-3′; GSP3, 5′-GAC GCG CAA GAC ATT GTG AG-3′) for the first and second PCR respectively. The amplified fragment was cloned into plasmid pMD18-T (TaKaRa, Dalian, China) and analyzed by automated sequencing (Sangon, Shanghai, China).

Xia L.Q., Chen J.L., Zhang H.L., Cai J., Zhou S, & Lu Y.S. (2019). Identification of virion-associated transcriptional transactivator (VATT) of SGIV ICP46 promoter and their binding site on promoter. Virology Journal, 16, 110.

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5' RACE for cDNA Amplification

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5′-RACE was performed essentially as described in the Invitrogen 5′-RACE system for Rapid Amplification of cDNA ends version 2.0 manual, with only minor modifications. Briefly, cDNA was generated using gene-specific primers (dnaK_GSP1) and the iScript cDNA Synthesis kit from Bio-Rad according to the supplier's protocol. cDNA was purified using the Qiagen nucleotide removal kit and a poly-C tail was added using the TdT enzyme (Invitrogen™) and dCTP. 5′-RACE PCR was performed using nested primers located 5′ to dnaK_GSP1 (dnaK_GSP2 or dnaK_GSP3) and the 5′-RACE Abridged Anchor Primer or Abridged Universal Amplification Primer (AUAP) described in the Invitrogen manual. 5′-RACE products were gel purified using the Qiagen gel extraction kit and sequenced at the University of Florida ICBR core facilities.

Palmer S.R, & Burne R.A. (2015). Post-transcriptional regulation by distal Shine-Dalgarno sequences in the grpE-dnaK intergenic region of Streptococcus mutans. Molecular microbiology, 98(2), 302-317.

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Profiling miR-9 Transcripts in Zebrafish

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Total RNA from 24 h post-fertilization (hpf) embryos was extracted using Trizol (Ambion), treated with DNase I (Promega) and reverse transcribed with Superscript II reverse transcriptase (Invitrogen) using random hexamer primers. Seven paralogous copies of assembled primary transcripts (described below) of miR-9 were amplified by PCR using the following primers (Supplementary Table S2), and cloned using Strataclone PCR cloning kit (Agilent technologies). 5′ RACE PCR experiments were performed using the 5′ RACE system for rapid amplification of cDNA ends (Invitrogen), using the listed primers (Supplementary Table S2). Antisense DIG labeled RNA probes for each miR-9 transcripts were synthesized and in situ hybridization performed using standard protocols (36 (link)).

Nepal C., Coolen M., Hadzhiev Y., Cussigh D., Mydel P., Steen V.M., Carninci P., Andersen J.B., Bally-Cuif L., Müller F, & Lenhard B. (2015). Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs. Nucleic Acids Research, 44(7), 3070-3081.

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5' End Mapping of hlyII Operon

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The 5′ end of the hly_II operon transcript was determined using the 5′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen). Total RNA was isolated from cultures of the J96 strain grown in LB at 37 °C up to an OD_600 nm of 1.0. Retrotranscription was done using the oligo hlyIIBamIII, specific for the hly_II operon. Next, a polyC tail was added to the 3′ end of the cDNA by using terminal deoxynucleotidyl transferase. Two rounds of PCR amplification were performed using specific primers for the hly_II operon, hlyII-GSP2 and hlyII-GSP3, and primers provided by the commercial kit. The final PCR product was isolated and sequenced.

Velasco E., Wang S., Sanet M., Fernández-Vázquez J., Jové D., Glaría E., Valledor A.F., O’Halloran T.V, & Balsalobre C. (2018). A new role for Zinc limitation in bacterial pathogenicity: modulation of α-hemolysin from uropathogenic Escherichia coli. Scientific Reports, 8, 6535.

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lacZ Gene Expression in E. coli

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Total RNA from E. coli MG1656/pPintI2-1 (Table 1) was extracted and cDNA specific to the lacZ gene was synthetized by a reverse transcriptase (TaKaRa) using primers 30, 31, and 32 (Supplementary Table S1). After purification, cDNA was used as template for the 5′RACE experiment in accordance with the manufacturer’s recommendations (5′RACE System for Rapid Amplification of cDNA Ends, Invitrogen), and using the TaKaRa Ex Taq^TM DNA polymerase (TaKaRa Biotechnology). The purified PCR product was cloned in the pGEM^®-T Easy vector (Promega) in E. coli DH5aaa and nine clones were sequenced.

Jové T., Da Re S., Tabesse A., Gassama-Sow A, & Ploy M.C. (2017). Gene Expression in Class 2 Integrons Is SOS-Independent and Involves Two Pc Promoters. Frontiers in Microbiology, 8, 1499.

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