For transcriptional reporter assays, HUVECs cells were transfected with p75NTR proximal promoter or miR-503 promoter (Switchgear Genomics) constructs using GenJet™ In Vitro DNA Transfection Reagent (SignaGen) and 24hrs later, cells were exposed to L-Glucose or D-Glucose or tranduced with Ad.p75 (Ad.Null as control) for 24h and lysed using buffer supplied with the Dual-Luciferase Reporter Assay System (Promega). Mutated plasmids were generated using GeneTailor Mutagenesis system kit (Invitrogen). To investigate whether miR-503 directly regulates VEGFA, and EFNB2 expression, portions of the 3′-UTR of these potentials target genes were inserted downstream of a luciferase open reading frame (pLUC). VEGFA 3′-UTR (S204537) and EFNB2 3′-UTR (S213182) vectors were purchased from SwitchGear Genomics. Vectors in which five nucleotide mutations were inserted in the 3′-UTR sequences (VEGFA: 293-299; EFNB2: 1126-1132) complementary to the miR-503 “seed” sequence were prepared using GeneTailor kit (Invitrogen). Primers are listed in Supplementary Table 1 . Luciferase constructs were transfected into HEK293T cells together with either pre-miR-503 or a scrambled oligonucleotide sequence (control). Cells were cultured for 48h and assayed with the Dual-Luciferase Reporter Assay System (Promega).
Genetailor kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneTailor kit is a laboratory equipment product developed by Thermo Fisher Scientific. It is designed for genetic engineering applications, facilitating the precise modification of DNA sequences. The core function of the GeneTailor kit is to enable efficient and accurate gene editing through a streamlined workflow.
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Lab products found in correlation
5 protocols using genetailor kit
1
Transcriptional Reporter Assay Protocol
2
Generation of Mouse-Adapted Influenza A/CA04/09 (H1N1) Virus
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A/California/04/09 (CA04, H1N1) split vaccine antigen was kindly provided by The Green Cross Reference Lab (Yongin, Korea). Mouse-adapted influenza A/California/04/09 (maCA04, H1N1) virus was generated by site-directed mutagenesis using the GeneTailor kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, the positions at 12, 58, and 88 in PB1-F2 stop codons, which are required for PB1-F2 protein expression, were mutated to serine (S), tryptophan (T), and tryptophan (T), respectively. In addition, the previously known PA (T97I) mutant was also incorporated to enhance virulence in mice [20 (link)]. The resulting maCA04 (H1N1) virus was propagated in 11-day-old-embryonic eggs at 37℃ for 72 hours. The allantoic fluid containing the virus was harvested and centrifuged to remove unwanted debris at 3,000 rpm for 10 minutes at 4℃. The harvested virus was dispensed in aliquots and stored at -80℃ until further use. Virus titers were calculated as previously described [20 (link),21 (link)].
3
Validating miR-532-5p's Regulation of BACH1
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To investigate whether miR-532-5p directly regulates BACH1 expression, portions of the 3′ UTR of the potential target gene was inserted downstream of a luciferase open reading frame (pLUC). BACH1 3′ UTR vector was purchased from SwitchGear Genomics. Vectors in which five nucleotide mutations were inserted in the 3′ UTR sequences (position 2,982–2,988 of BACH1 3′ UTR) complementary to the miR-532 “seed” sequence were prepared using GeneTailor kit (Invitrogen). Primers are as follows: forward, 5′-TTTAAATGTTTTATTttaattaGTAATAAACTAT-3′, and reverse, 5′-AATTTTCATTCTACCCAACAAGTTTC-3′. Luciferase constructs were transfected into HEK293T cells together with pRenilla vector and either miR-532-5p mimic or a scrambled oligonucleotide sequence (control). Cells were cultured for 48 hr and assayed with the Dual-Luciferase Reporter Assay System (Promega).
4
Transcriptional Regulation of p75NTR and miR-503
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For transcriptional reporter assays, HUVEC cells were transfected with p75NTR proximal promoter or miR-503 promoter (Switchgear Genomics) constructs using GenJet In Vitro DNA Transfection Reagent (SignaGen), and, 24 h later, cells were exposed to L-Glucose or D-Glucose or transduced with Ad.p75 (Ad.Null as control) for 24 h and lysed using buffer supplied with the Dual-Luciferase Reporter Assay System (Promega). Mutated plasmids were generated using GeneTailor Mutagenesis system kit (Invitrogen). To investigate whether miR-503 directly regulates VEGFA, and EFNB2 expression, portions of the 3′UTR of these potentials target genes were inserted downstream of a luciferase open reading frame (pLUC). VEGFA 3′UTR (S204537) and EFNB2 3′UTR (S213182) vectors were purchased from SwitchGear Genomics. Vectors in which five nucleotide mutations were inserted in the 3′UTR sequences (VEGFA: 293–299; EFNB2: 1,126–1,132) complementary to the miR-503 ‘seed' sequence were prepared using the GeneTailor kit (Invitrogen). Primers are listed in Supplementary Table 1 . Luciferase constructs were transfected into HEK293T cells together with either pre-miR-503 or a scrambled oligonucleotide sequence (control). Cells were cultured for 48 h and assayed with the Dual-Luciferase Reporter Assay System (Promega).
5
Site-directed Mutagenesis of IR4 Gene
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Site-directed mutations were generated using the Invitrogen GeneTailor kit (Invitrogen; Carlsbad, CA, USA), which allows the use of a mutagenic oligonucleotide primer to produce the desired mutation from cloned DNA that has been previously methylated. The procedure also incorporated PCR amplification, and after the introduction of the reaction products into E. coli DH5α, unmutated template plasmid was degraded by the McrBC endonuclease that digested methylated DNA, allowing the efficient recovery of the amplified (mutated) plasmid. IR4 was cloned between the HindIII-EcoRI sites of pBlueScript SK II+ and used for mutagenesis, after which the mutations were confirmed by sequencing. IR4 variants were then amplified using forward and reverse primers with engineered ClaI tags, digested with ClaI and ligated to the BgaH reporter plasmid pRV2 (see above).
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