Amplified fragments with PEDV-M-F/R, PoRV-NSP5-F/R, or PDCoV-N-F/R were synthesized and cloned into the pUC57 vector by Genewiz (Suzhou, China). The plasmid was then used as the standard positive control. The concentration of the plasmid was converted to copy number using the following formula: y (copies/μL) = (6.02 × 1023) × (x(ng/μL) × 10−9 DNA)/(DNA length × 660) [22 (link)]. The 10-fold serially diluted standard plasmids (6.0 × 101–6.0 × 106 copies/μL) were used as templates to generate the standard curve of the triplex real-time RT-PCR assay.
Puc57 vector
Manufactured by Genewiz
Sourced in China
The PUC57 vector is a plasmid commonly used for gene cloning and expression in Escherichia coli. It contains the pMB1 origin of replication and the ampicillin resistance gene for selection purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Plvx tre3g vector, by Takara Bio (2 mentions)
Quickchange lightning site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Psicheck 2 plasmid, by Promega (1 mentions)
Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Topo cloning kit, by Thermo Fisher Scientific (1 mentions)
Sanger sequencing, by Eurofins (1 mentions)
Vehicle control, by Merck Group (1 mentions)
Abiraterone, by Selleck Chemicals (1 mentions)
Spectramax id3, by Molecular Devices (1 mentions)
Nef832001ea, by PerkinElmer (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Dig rna labeling mix, by Roche (1 mentions)
9 protocols using puc57 vector
1
Triplex RT-PCR Assay for Porcine Coronaviruses
2
Engineered TPNOX Variants for Mammalian Cells
3
Luciferase Reporter Assay for miR-302d Targeting
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The full-length wild-type 3′ UTR of p53 was chemically synthesized and cloned into the pUC57 vector (Genewiz). The wild-type 3′ UTR of p53 was then subcloned into the pSiCheck2 plasmid (Promega) at XhoI and NotI sites to generate the WT-Luc reporter. Mutations at the miR-302 recognition site were introduced by PCR-based site-directed mutagenesis, as described previously (Zhao et al., 2014 (link)) to generate the Mut-Luc reporter. To generate the 302-Luc reporter, oligonucleotides complementary to miR-302d were ligated into the pSiCheck2 plasmid at XhoI and NotI sites. Oligonucleotides used for luciferase reporter construction are listed in Table S4 . All constructs were confirmed by Sanger sequencing. Forty-eight hours before the luciferase assay, 3 × 103 HEK293T cells were transfected with 5 ng of luciferase reporters and 3 pmol of miR-302d mimic or control mimic conjugated with Dy547 (C-310372-05 and CP-004500-01, GE Dharmacon) by Lipofectamine 2000 (Thermo Fisher Scientific). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System as per the manufacturer’s instructions (Promega) on a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek).
4
Overexpression of miR-590 and TGFβR2
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To overexpress the miR-590, the 81 base pairs (bp) genomic sequence, which encodes mature miR-590, was cloned into the PUC57 vector (manufactured by Genewiz, Inc., South Plainfield, NJ, USA). The sequence used was: 5′-AGTCAGAAATGAGCTTATTCATAAAAGTGCAGTATGGTGAAGTCAATCTGTAATTTTATGTATAAGCTAGTCTCTGATTGA-3′. Subsequently, the premature sequence was transferred into pcDNA3.1 (Invitrogen Life Technologies) downstream of the cytomegalovirus (CMV) promoter between the restriction sites HindIII and EcoRI. For the luciferase reporter plasmids, the 3′ UTR of the mouse TGFβR2, or its mutant variations, was amplified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) from the mouse genome, and inserted downstream of the luciferase coding sequence of the pcDNA3.1-luciferase reporter plasmid between the restriction sites BamHI and EcoRI. For the overexpression of TGFβR2, the coding sequence was cloned to generate the plasmid pCMV-tag-2B-TGFβR2 using the restriction sites of BamHI and Xhol, and (forward: 5′-CGCGGATCCCTGTCCACTTGCGACAACCAG-3′ and reverse: 5′-GGCCTCGAGTTTGGTAGTGTTCAGCGAGC-3′). All the plasmids used in the present study were verified by sequencing.
5
Engineered TPNOX Variants for Mammalian Cells
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A synthetic TPNOX gene cloned into pUC57 vector was obtained from Genewiz (See Supplementary Fig. 1 ). TPNOX-encoding gene was digested with BamHI and XhoI and subcloned into a pET30a vector (Novagen). LbNOX variants D177A/A178R/P184R, D177A/A178R/M179H/P184R and D177A/A178R/M179S/P184R were obtained using a Quickchange Lightning site-directed mutagenesis Kit (Agilent) using pET30a plasmid containing TPNOX sequence as a template and the following primers (only sense sequences are shown):
For expression in mammalian cells synthetic TPNOX and mitoTPNOX genes cloned into pUC57 vector were obtained from Genewiz (SeeSupplementary Fig. 1 ) and were subsequently subcloned into a pLVX-TRE3G vector (Clontech) using NotI and XhoI restriction cites.
For expression in mammalian cells synthetic TPNOX and mitoTPNOX genes cloned into pUC57 vector were obtained from Genewiz (See
6
Wheat TILLING Mutant Library Generation
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TILLING mutations in the NAM-A1 gene (TraesCS6A02G108300) were identified using the TILLING database at www.wheat-tilling.com [28 (link)]. Specific alleles were selected based on conservation of the residue in the NAC domain (PSSM viewer, pfam02365) and predicted SIFT scores [59 (link)]. For simplicity, the Kronos mutant lines are labelled as ‘K’ followed by their four-digit identifier (e.g. Kronos 2711 is referred to as K2711).
The NAM-A1 sequence from T. aestivum (Transcript TraesCS6A02G108300.2) was synthesized into the pUC57 vector (Genewiz) and cloned into the pCR8 vector using the TOPO cloning kit (ThermoFisher Scientific) using primers in Additional file2 : Table S4. Site-directed mutagenesis was carried out using the primers in Additional file 2 : Table S5, to acquire the allelic series of NAM-A1 variants. All cloned products were confirmed using Sanger sequencing (Eurofins Genomics). The NAM-B1 sequence was obtained from T. turgidum subsp. dicoccoides (GenBank accession DQ869673.1) and was synthesized as detailed above (Additional file 3 ).
The NAM-A1 sequence from T. aestivum (Transcript TraesCS6A02G108300.2) was synthesized into the pUC57 vector (Genewiz) and cloned into the pCR8 vector using the TOPO cloning kit (ThermoFisher Scientific) using primers in Additional file
7
Characterization of SLCO1B3 3'UTR Regulation
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The SLCO1B3 3′UTR was synthesized with flanking NotI and XhoI restriction sites in a pUC57 vector by GENEWIZ, Inc (South Plainfield, NJ). The synthesized sequence was then cloned into complementary restriction sites in a psiCHECK-2 vector, which contains both renilla and firefly luciferase reporter genes (Promega, CAT#: C8021). The constructed plasmid was sequenced to verify successful cloning.
22Rv1 cells were seeded in a 96-well dish for 24 h before transfection with 100 ng of psiCHECK-2 empty vector or 3′UTR plasmid using Lipofectamine 2000 (Life Technologies, Cat#: 11668019). The following day, cells were treated with 20 µM abiraterone (SelleckChem, Cat#: S1123) or vehicle control (0.4% DMSO) (Sigma Aldrich). After a 24-h treatment period, samples were lysed and assayed using the Dual-Luciferase Reporter Assay System (Promega, Cat#: E1910) per manufacturer’s protocol. Raw luminescence was measured for Renilla and firefly reporters using SpectraMax iD3 (Molecular Devices). For microRNA mimic treated samples, cells were instead co-transfected for 24 h with the psiCHECK-2 plasmid or empty vector and 1 pmol of hsa-miR-579-3p miRvana mimic or negative control (Life Technologies, Cat#: 4464066) before performing the reporter assay.
22Rv1 cells were seeded in a 96-well dish for 24 h before transfection with 100 ng of psiCHECK-2 empty vector or 3′UTR plasmid using Lipofectamine 2000 (Life Technologies, Cat#: 11668019). The following day, cells were treated with 20 µM abiraterone (SelleckChem, Cat#: S1123) or vehicle control (0.4% DMSO) (Sigma Aldrich). After a 24-h treatment period, samples were lysed and assayed using the Dual-Luciferase Reporter Assay System (Promega, Cat#: E1910) per manufacturer’s protocol. Raw luminescence was measured for Renilla and firefly reporters using SpectraMax iD3 (Molecular Devices). For microRNA mimic treated samples, cells were instead co-transfected for 24 h with the psiCHECK-2 plasmid or empty vector and 1 pmol of hsa-miR-579-3p miRvana mimic or negative control (Life Technologies, Cat#: 4464066) before performing the reporter assay.
8
In situ Hybridization of lncRNA in Mouse Retina
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The lncRNA probe templates, with the T3 and T7 adaptors, were firstly synthesized and inserted in pUC57 vector (Genewiz, Suzhou, China) and then amplified with the M13 primers by PrimeSTAR HS DNA Polymerase (Takara, Code No. R010A) (see Additional file 8 : File S1 for probe sequences). Sense and antisense of probes were transcribed with T7 and T3 RNA polymerases, respectively. DIG RNA labeling mix (Roche, 1,277,073) was used to label the probes. Probes of Pou4f1, Pou4f2 and Pou4f3 were used as the positive control, which were generated according to the method described previously [42 (link)].
Retinas from mice at E15.5 were used for detection of gene expression. The embryos at E15.5 were fixed in 4% paraformaldehyde in PBS at 4 °C for 6 h and dehydrated in 30% sucrose in PBS at 4 °C. After dehydration, the embryos were embedded in OCT (ThermoFisher Thermo NEG50, cat. no. 6506) at − 80 °C. FISH on sectioned tissues were carried out according to the previously described method [43 (link)]. Probes were detected by using Anti-digoxigenin-HRP (1:1000) (Perkin Elmer, cat. no. NEF832001EA) and Tyramide Signal Amplification system (Perkin Elmer, TSA Plus, cat. no. NEL744001KT) according to the manufacturer’s recommendation.
Retinas from mice at E15.5 were used for detection of gene expression. The embryos at E15.5 were fixed in 4% paraformaldehyde in PBS at 4 °C for 6 h and dehydrated in 30% sucrose in PBS at 4 °C. After dehydration, the embryos were embedded in OCT (ThermoFisher Thermo NEG50, cat. no. 6506) at − 80 °C. FISH on sectioned tissues were carried out according to the previously described method [43 (link)]. Probes were detected by using Anti-digoxigenin-HRP (1:1000) (Perkin Elmer, cat. no. NEF832001EA) and Tyramide Signal Amplification system (Perkin Elmer, TSA Plus, cat. no. NEL744001KT) according to the manufacturer’s recommendation.
9
Genetic Manipulation with Synthetic Nucleic Acids
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Synthetic nucleic acids were purchased from Integrated DNA Technologies (IDT), GenScript or Genewiz. For knock-in of a single split-wrmScarlet11 or sfGFP11 sequence, 200-mer HDR templates were ordered in ssODN form (synthetic ssODN) from IDT. For knock-in of split-wrmScarlet11 repeats, HDR templates were ordered in dsDNA form (plasmids) from GenScript or Genewiz. For plasmids injected as extrachromosomal arrays, sequences were synthesized and cloned into the pUC57 vector (Genewiz). The complete set of crRNAs and DNA sequences and plasmids used for the experiments described here can be found in Supplementary Tables S1, S4, and S5.
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