All the receptors used in this study were either cloned into bicistronic pEFIN3 vector (Euroscreen) for stable expression or pcDNA3 vector (Life Technologies) for transient expression (Table S1 ). All constructs were verified by sequencing prior to transfection.
Pcdna3 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Canada, France
The pcDNA3 vector is a commercially available plasmid commonly used for gene expression in mammalian cells. It contains a cytomegalovirus (CMV) promoter for high-level expression of the gene of interest and a neomycin resistance gene for selection of transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (30 mentions)
Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (7 mentions)
Pcdna3, by Thermo Fisher Scientific (6 mentions)
Dual luciferase reporter assay system, by Promega (5 mentions)
Pegfp n1 vector, by Takara Bio (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Gene pulser xcell electroporation system, by Bio-Rad (3 mentions)
Pgl3 basic vector, by Promega (3 mentions)
Viromer red, by Biozym (3 mentions)
Pegfp c2, by Takara Bio (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (3 mentions)
Pegfp c1 vector, by Takara Bio (3 mentions)
Pgl3 vector, by Promega (3 mentions)
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Ecori, by New England Biolabs (2 mentions)
Pci vector, by Promega (2 mentions)
212 protocols using pcdna3 vector
1
Stable and Transient Receptor Expression
2
Identification of LncRNA-HIT and U1 Interacting Proteins
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Mouse LncRNA-HIT and U1 genes were cloned into the pCDNA3 vector (Life Technologies, Grand Island, NY) and transcribed using T7 RNA polymerase and biotin-16-UTP as described by the manufacturer (Roche, Indianapolis, IN). Cell lysates from E11.0 wild type embryos limbs which were homogenized in mRIPA buffer and centrifuged as described [103 (link)]. 1 mg of total protein was incubated with either the LncRNA-HIT or U1 transcripts tagged with biotin-16-UTP 12 H at 4°C. Following incubation, the RNA-protein mixtures were incubated with 100 μl prewashed streptavidin beads for 2.5 H at 4°C. After incubation, the streptavidin beads were washed five times in ice cold PBS and combined with 50 μl of an SDS elution buffer containing 125 mM Tris/HCl pH 6.8, 20% glycerol, 4% SDS, 10% 2-mercaptoethanol and 0.02% bromphenol blue. After two rounds of elution, the recovered protein mixture was heated to 95°C for 5 minutes and fractionated by SDS-PAGE electrophoresis using a 4–12% gradient gel (Nu Page, Invitrogen). The gels were stained with Coomassie blue and the individual bands digested with trypsin and submitted for identification by mass spectroscopy by the OHSU Proteomics Core Facility as described [104 (link)].
3
Cloning and Expression of PPAR-gamma2 cDNA
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The expression plasmid for full-length PPARγ2 cDNA was cloned from a cDNA library of human adipocyte [20 (link)] and inserted into a pcDNA3 vector (Life Technologies, Carlsbad, CA) with a FLAG tag sequence. Anti-PPARγ antibody (#sc-7273) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Troglitazone (Sigma, St. Louis, MO) was dissolved in DMSO.
4
Podoplanin cDNA Expression in CHO Cells
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The amplified human podoplanin cDNA was subcloned into a pcDNA3 vector (Life Technologies) and a FLAG epitope tag was added at the C-terminus. Substitution of amino acids to alanine in podoplanin was performed using a QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA).(30 (link),32 (link)) CHO-K1 cells were transfected with the plasmids using a Gene Pulser Xcell electroporation system (Bio-Rad Laboratories).
5
Cloning and Tagging Anti-Coronavirus scFv
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All new plasmids used are cloned into pcDNA3 vector (Life Technologies). The 2A-P*, 2A*, 2A(M) with mutated codons, T2A, CD99L2, POTE, TBCA, SPIRE2 and C-TAIL coding sequences were inserted by digestion with enzymes KpnI and EcoRI (NEB) in frame at the C-terminus of previously described scFv anti-coronavirus 6A.C3 mAb [60] (link), using oligoes described in Supplementary Table 3. When indicated, SV5-tag (GKPIPNPLLGLD), roTag (SISSSIFKNEG) [61] (link), HA-tag (YPYDVPDYA) and N-glycosylation site (NGT) were engineered at the indicated positions by site-directed mutagenesis (QuikChange Site Directed Mutagenesis Kit; Stratagene). Plasmids encoding secretory proteins bear an immunoglobulin secretion leader peptide [62] (link) at the N-terminus as previously described [61] (link). MHC-Iα having C-terminal 2A* and 2A-P* sequence was derived from a previously described MHC-Iα plasmid [39] (link). The coding sequence of BioID2 was previously described [42] (link). Plasmids expressing cyt-BirA and BiP mutant T37G were previously described [35] (link). CCHFV-L OTU plasmid was kindly provided by Adolfo García-Sastre, the N-terminal FLAG-tagged human YOD1-C160S plasmid by Christian Schlieker, and the His6-tagged rat p97QQ plasmids by Linda Hendershot.
6
Cloning and Mutagenesis of HIV-1 Vpr
7
Construction of H2AX-GFP Reporter Construct
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A 1425-bp fragment containing the H2AX gene promoter was amplified from mouse genomic DNA. This fragment was used as a template for a nested PCR (1040-bp product), aimed at introducing the NdeI and NheI restriction sites at the distal and proximal ends of the H2AX promoter, respectively (all primers are given in Supplementary Table 1 ). Restriction of this product with the above enzymes resulted in a 1003-bp fragment which was inserted into the AseI–NheI sites of the pEGFP-C3 vector (Clontech, Mountain View, CA). From this vector, a 1968-bp H2AX–GFP, KpnI–XhoI, fragment was excised and subcloned into the respective sites of the pCDNA3 vector (Life Technologies, Carlsbad, CA). The resulting chimeric H2AX–GFP fragment consisted of 960 bp from the H2AX promoter (including its TATA-box at the original positions: −61 to −64 relative to ATG), followed by a 6-bp common NheI restriction sequence, 11-bp sequences upstream of the EGFP start codon and the full EGFP coding region terminated by 263 bp of the 3′-untranslated region, including two poly(A) sites.
8
Noncanonical NF-κB Regulation of Spib Promoter
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The coding sequences of mouse RelB and p52 (Nfkb2) were amplified from FAE-derived cDNA and subcloned into pcDNA3 vector (Life Technologies). The putative Spib promoter region (approximately −1,930 to 0) was amplified from a bacterial artificial chromosome (BAC) clone (RP23-455C13 purchased from Life Technologies), and subcloned into pGL3Basic vector (Promega). To determine the direct interaction of noncanonical NF-κB and Spib promoter, mICcl2 (kindly provided by Dr. Jean-Pierre Kraehenbuhl; Bens et al., 1996 (link)) cells were transfected with the combination of these expression and luciferase reporter vectors by Lipofectamine 2000 (Life Technologies). After the overnight culture, cells were lysed. Then luciferase activity was measured by the dual-luciferase reporter assay system (Promega).
9
In vivo Electroporation of Chick Embryos
10
Overexpression and Knockdown of SRF
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The human SRF expression plasmid was kindly provided by Dr. Eric Olson (University of Texas Southwestern Medical Center). The pcDNA3-CDH1 plasmid was constructed as previously described43 (link) by inserting the CDH1 cDNA into the pcDNA3 vector (Life Technologies) at the Xhol and Xbal sites. Transfections were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. siRNA, miRNA mimic, and miRNA inhibitor transfection were performed following the same procedure. The sense strand sequences of SRF siRNAs designed to target human cells were as follows: SRF siRNA no. 1, 5′-CCACAACAGACCAGAGAAUTT-3′ SRF siRNA no. 2, 5′-GU UCCUGACAGCAUCAUCUTT-3′ SRF siRNA no. 3, 5′-CCCUGUUUCAGCAGUUCAGTT-3′. Successful overexpression and knockdown of SRF were confirmed by western blot (Supplementary Figure S1 ). The miR-199a-5p mimic (miR-199a-5p), miR-199a-3p mimic (miR-199a-3p), miRNA mimic negative control, miR-199a-5p inhibitor, miR-199a-3p inhibitor, and miRNA inhibitor negative control oligonucleotides were chemically synthesized and purified by RiboBio (Ribobio).
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