To construct pMcp-1.BDNF and pMcp-1.NRF2 vectors, a fragment of the murine Mcp-1 promoter (560 bp immediately upstream of the initiation codon ATG; accession number U12470 ) and BDNF cDNA (KIBB9810; Promega) or NRF2 cDNA (KIBB7828; Promega) were inserted into a pAAV-MCS Promoterless Expression Vector (Cell Biolabs). To construct pMcp-1.NRF2-2A-EGFP, the same fragment of the Mcp-1 promoter, NRF2 cDNA, linker peptide 2A cDNA, and EGFP cDNA (Clontech) were inserted into the pAAV-MCS Promoterless Expression Vector using the Gibson assembly system (New England Biolabs). To construct pCMVNRF2-2A-EGFP, Nrf2 cDNA, 2A peptide, and egfp cDNA (Clontech) were inserted into a pAAV-CMVp-MCS Expression Vector (Cell Biolabs) using the Gibson assembly system (New England Biolabs). For pAAV.CMV.EGFP using pAAV-GFP vector (Cell Biolabs), viral vectors AAV2/2 were generated and purified following a method described previously.48 (link) Each vector (1.0 × 1012 genome copy [gc]/mL) was injected at 2.0 μL per injection into the vitreous of an anaesthetized mouse. The dose was the same in all experiments involving intravitreal injection of AAV in this study.
Gibson assembly system
Manufactured by New England Biolabs
Sourced in United States, Japan
The Gibson Assembly system is a molecular cloning technique used to assemble multiple DNA fragments into a single construct. It enables the seamless joining of DNA sequences without the need for restriction enzymes or ligase. The system relies on the exonuclease, polymerase, and ligase activities of the T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase to create overlapping regions between the DNA fragments, allowing them to be assembled in a single, isothermal reaction.
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Lab products found in correlation
Phage puro, by New England Biolabs (2 mentions)
Paav gfp vector, by Cell Biolabs (1 mentions)
Paav mcs promoterless expression vector, by Cell Biolabs (1 mentions)
Egfp cdna, by Takara Bio (1 mentions)
Puc19 plasmid, by Takara Bio (1 mentions)
Pentr d topo cloning kit, by Thermo Fisher Scientific (1 mentions)
Lsm 880 axioobserver, by Zeiss (1 mentions)
Pei max, by Polysciences (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
Pet15b, by Merck Group (1 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Pei reagent, by Polysciences (1 mentions)
Pgl3 pro, by New England Biolabs (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Kod plus neo, by Toyobo (1 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
Kod onetm pcr master mix, by Toyobo (1 mentions)
Mach1t1r, by Thermo Fisher Scientific (1 mentions)
15 protocols using gibson assembly system
1
Constructing Targeted Gene Expression Vectors
2
Engineered FLNC Protein with V2297M
3
Visualizing OsRZF1 expression in rice protoplasts
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The ORF of the OsRZF1 gene without the stop codon was amplified (Supplemental Table S1 ) and cloned to the entry vector with a pENTR™/D-TOPO® Cloning Kit (Invitrogen, USA). Then the 2 × 35 s promoter (Marquès-Bueno et al., 2016 (link)) and amplified OsRZF1 ORF fused with EGFP marker were inserted into the pUC19 plasmid (Takara Bio Inc. Japan) using a Gibson assembly system (New England BioLabs, USA). Rice protoplasts were isolated and transformed according to the method previously reported (Saito et al. 2013 (link)). After 18 h, the EGFP fluorescence was detected using Zeiss LSM880 AxioObserver, equipped with an Argon laser as the excitation source, through a 20x/0.8 plan-Apochromat objective lens. EGFP was excited with an Argon laser at 561 nm and an intensity of 0.5% and detected between 597–624 nm with a detector gain value of 900. Images were analyzed and merged by ImageJ (NIH).
4
Cloning and Expression of Dectin-1 CRD
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We cloned cDNA encoding the carbohydrate recognition domain (CRD) of mouse dectin-1 from BMDCs from C57BL/6J mice using primers pDectin1-CRD-Fw1 (5′-TTGCACTAAGTCTTGCACTTGTCACatgccttcctaattggatcatgcatg-3′ ) and pDectin1-CRD-Rv1 (5′-GCTTACAACCACAATCCCTGGGCACcagttccttctcacagatactgtat-3′ ). The pCAG-Neo mIgG1-Fc plasmid (FUJIFILM Wako Pure Chemical Corp.) was digested with XhoI–SpeI, and the cDNA fragment was inserted using the Gibson Assembly System (New England Biolabs Inc.) to construct the expression vector pmDectin-1_mIgG1-Fc. Our dectin-1 CRD cDNA sequence was identical to that of Clec7a-202, as recorded in the Ensembl database (http://www.ensembl.org/index.html , transcript ID: ENSMUST00000184581.2).
The expression vector pmDectin-1_mIgG1-Fc was transfected into HEK293T cells using PEI-MAX (Polysciences Inc.) or GenePORTER2 (Gelantis Inc.), and cells were cultivated in RPMI 1640 medium with 10% (w/v) FBS for 2 days at 37°C, 5% CO2 to generate Fc dectin-1 in the culture supernatant. Culture supernatant was stored at −80°C prior to use in the Fc dectin-1 deposition assay.
Fc dectin-2 was purchased from Enzo Life Science, Inc., was reconstituted with sterile water, and was stored at −80°C.
The expression vector pmDectin-1_mIgG1-Fc was transfected into HEK293T cells using PEI-MAX (Polysciences Inc.) or GenePORTER2 (Gelantis Inc.), and cells were cultivated in RPMI 1640 medium with 10% (w/v) FBS for 2 days at 37°C, 5% CO2 to generate Fc dectin-1 in the culture supernatant. Culture supernatant was stored at −80°C prior to use in the Fc dectin-1 deposition assay.
Fc dectin-2 was purchased from Enzo Life Science, Inc., was reconstituted with sterile water, and was stored at −80°C.
5
Construction of IFT20-4xFKBP12 Lentivirus
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Four tandem repeats (4xFKBP) of the FK506 binding protein were PCR amplified from a vector containing IFT20-4xFKBP12 (gift of T. Inoue). IFT74-3xFlag was amplified from TE60. Both fragments were inserted in the lentiviral vector pHAGE-puro with the Gibson Assembly system (New England Biolabs).
6
Lentiviral Expression of IFT20-4xFKBP12
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Four tandem repeats (4xFKBP) of the FK506 binding protein and IFT20-3xFlag fragments were PCR amplified from a vector containing IFT20-4xFKBP12 (gift of T. Inoue). Both fragments were inserted in the lentiviral vector pHAGE-puro with the Gibson Assembly system (New England Biolabs).
7
CEPDL1/2 ORF Cloning and Arabidopsis Transformation
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We cloned the CEPDL1 or CEPDL2 ORFs into XbaI-, SacI-digested pBI121 vector downstream of the CaMV 35 S promoter using the Gibson Assembly system (New England Biolabs). General sequence analysis was performed using GENETYX-MAC software (Genetyx, Tokyo, Japan). Transgenic Arabidopsis plants were generated by the standard Agrobacterium-mediated transformation using floral dip method. The primer list is shown in Supplementary Data 2 .
8
Generating MVA-CoV2-BMEP Recombinant Virus
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CoV2-BMEP construct was synthesized and subcloned into the pcDNA vector backbone by GeneArt (Thermo Fisher Scientific, Waltham, MA, USA), resulting the plasmid pcDNA-CoV2-BMEP (shortly DNA-CoV2-BMEP). The plasmid transfer vector pCyA20-CoV2-BMEP was obtained by Gibson assembly system (New England Biolabs, Ipswich, MA, USA) according to manufacturer’s instructions and using pCyA20 (58 (link)) and DNA-CoV2-BMEP plasmids as templates. The sequence of the resulting plasmid was confirmed by PCR and DNA sequence analysis (Macrogen, Seoul, South Korea). The pCyA20-CoV2-BMEP (8579 bp) plasmid was used for the generation of MVA-CoV2-BMEP recombinant virus allowing the insertion of CoV2-BMEP construct into the viral TK locus of MVA-WT.
9
Cloning and Validation of Fungal Genes
10
Cloning and Mutation Analysis of Arabidopsis Genes
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The sequences of the exon-intron-exon from AT5G56870, AT1G08450, and AT3G08930 were cloned into expression vector inter2 using Gibson Assembly system (NEB) with designed primers (Additional file 1 : Table S3). Corresponding mutations were introduced using Q5 Site-Directed Mutagenesis kit (NEB) with designed primers (Additional file 1 : Table S3) for AT5G56870. For AT1G08450 and AT3G08930, the exon-intron-exon sequence with designed mutations was synthesized (Sangon Biotech (Shanghai) Co., Ltd) and cloned into expression vector inter2. All constructs were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation. Transient expression was carried out in Nicotiana benthamiana according to the previous protocol [52 (link)]. RNA was extracted with RNeasy Plant Mini Kit (Qiagen), and semiquantitative RT-PCR was performed with designed primers (Additional file 1 : Table S3).
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