Pcdna3.1 v5 his topo ta

Manufactured by Thermo Fisher Scientific

Sourced in United States, China, France

The PcDNA3.1/V5-His TOPO TA is a laboratory tool used for the rapid cloning and expression of PCR products. It provides a quick and efficient method for the direct insertion of Taq polymerase-amplified DNA fragments into a plasmid vector for further analysis and study.

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Lab products found in correlation

Dulbecco s modified eagle s media, by Thermo Fisher Scientific (2 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions) Ecl kit, by Cytiva (1 mentions) Anti his antibody, by Abcam (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Pcdna4 hismax topo ta, by Thermo Fisher Scientific (1 mentions) Egfp c1, by Takara Bio (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Plasmid maxi kit, by Qiagen (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions)

9 protocols using pcdna3.1 v5 his topo ta

In vivo Transfection of YFP-LC32 and Fbxo21SMART-V5

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In vivo transfection experiments used the yellow fluorescent protein (YFP)–LC32 (link) and Fbxo21SMART-V5 plasmids. For the cloning of mouse Fbxo21/SMART gene, muscle cDNA was amplified by PCR using the primers forward: 3′-ACCATGGCGTCGGTAGCGGGGGACA-5′ and reverse: 3′-CTCGGCTGTGTCCTCCTTTGCACTG-5′.
The amplified sequence was cloned into pcDNA3.1/V5-His TOPO TA (Invitrogen) expression vector and sequenced. The list of the antibodies is described in Supplementary Table 2.
Uncropped blots are shown in Supplementary Fig. 19.

Milan G., Romanello V., Pescatore F., Armani A., Paik J.H., Frasson L., Seydel A., Zhao J., Abraham R., Goldberg A.L., Blaauw B., DePinho R.A, & Sandri M. (2015). Regulation of autophagy and the ubiquitin–proteasome system by the FoxO transcriptional network during muscle atrophy. Nature Communications, 6, 6670.

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Overexpression and detection of TLR2 protein

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ggTLR2-1 gene sequence encoding the full-length ORF were ligated into the mammalian expression vector, pcDNA3.1/V5-His-TOPO TA (Invitrogen, Shanghai, China). The resulting plasmid was introduced into HEK-293 cells by transfection using Lipofectamine®-2000 (Invitrogen). After 24 hours of transfection, cells were lysed with lysis buffer (1% Nonidet P-40, 10 mM EDTA, 1 mg/ml iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 50 mM Tris maleate, pH 8.6) at a density of 1 × 10⁷ cells/ml. Proteins of interest were separated by SDS-polyacrylamide gel electrophoresis [48 (link)], transferred to nitrocellulose membranes, and subjected to immunoblotting analysis with anti-His antibody (Abcam, Cambridge, UK). The antigen-antibody complex was detected using an ECL Kit (Amersham Pharmacia Biotech, Amersham, UK), following the manufacturer’s protocol.

Yong Y., Liu S., Hua G., Jia R., Zhao Y., Sun X., Liao M, & Ju X. (2015). Identification and functional characterization of Toll-like receptor 2–1 in geese. BMC Veterinary Research, 11, 108.

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Generation of ADAMTS13 Variants

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The ADAMTS13 wild-type (WT) cDNA was subcloned into the mammalian expression vector pcDNA^™3.1/V5-His TOPO^®TA (Invitrogen, Carlsbad, CA, USA). The nine ADAMTS13 variants (p.Val154Ile, p.Asp187His, p.Thr339Arg, p.Arg421Cys, p.Tyr603Cys, p.Asp836Gly, p.Arg925Gly, p.His1196Gln and p.Thr1249Pro) were separately introduced into the pcDNA3.1-ADAMTS13 WT expression vector by site-directed mutagenesis using PFU Turbo DNA polymerase (Stratagene, La Jolla, CA, USA) and specifically designed primers. The presence of each variant was confirmed by Sanger sequencing analysis.

Pagliari M.T., Lotta L.A., de Haan H.G., Valsecchi C., Casoli G., Pontiggia S., Martinelli I., Passamonti S.M., Rosendaal F.R, & Peyvandi F. (2016). Next-Generation Sequencing and In Vitro Expression Study of ADAMTS13 Single Nucleotide Variants in Deep Vein Thrombosis. PLoS ONE, 11(11), e0165665.

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Expression of Epitope-Tagged HAT Proteins

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Human embryonic kidney cells, HEK293T, and human cervical carcinoma cells, HeLa (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's media (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA). Full‐length human HAT cDNA in pCAGGS was generously provided by Dr. Eva Friebertshaeuser, Philipps University Marburg. The generation of human full‐length HATL‐5 cDNA in pcDNA 3.1/V5‐His TOPO^® TA (Invitrogen, Life Technologies, Grand Island, NY) in frame with a C‐terminal HIS‐tag and V‐5 epitope has previously been described (Miller et al., 2014 (link)). A control vector, pcDNA 3.1‐GFP, was used to assess transfection efficiency and as a negative control for western blotting. Transfection of cells was performed using Lipofectamine LTX according to the manufacturer's instructions (Invitrogen, Life Technologies, Grand Island, NY). Transfections were performed with 2.5 μg plasmid DNA.

Duhaime M.J., Page K.O., Varela F.A., Murray A.S., Silverman M.E., Zoratti G.L, & List K. (2016). Cell Surface Human Airway Trypsin‐Like Protease Is Lost During Squamous Cell Carcinogenesis. Journal of Cellular Physiology, 231(7), 1476-1483.

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Overexpression of Human HAT and HATL-5 in Cell Lines

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Human embryonic kidney cells, HEK293T, and human cervical carcinoma cells, HeLa (ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s media (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA). Full-length human HAT cDNA in pCAGGS was generously provided by Dr. Eva Friebertshaeuser, Philipps University Marburg. The generation of human full-length HATL-5 cDNA in pcDNA 3.1/V5-His TOPO^® TA (Invitrogen, Life Technologies, Grand Island, NY) in frame with a C-terminal HIS-tag and V-5 epitope has previously been described (Miller et al., 2014 (link)). A control vector, pcDNA 3.1-GFP, was used to assess transfection efficiency and as a negative control for western blotting. Transfection of cells was performed using Lipofectamine LTX according to the manufacturer’s instructions (Invitrogen, Life Technologies, Grand Island, NY). Transfections were performed with 2.5 μg plasmid DNA.

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Cloning and Mutating NAA80 Gene

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The NAA80 gene was inserted into vector pcDNA3.1/V5-His TOPO TA (Invitrogen), as we have described (7 (link)). The catalytically inactive mutant NAA80_CM (W105F, R170Q, G173D, and Y205F) and other mutants (Fig. 4D) were generated using the Q5 Site-Directed Mutagenesis Kit (NEB). The NAA80 constructs were cloned into vector pcDNA4/HisMax TOPO TA (Invitrogen), in which the Xpress tag was replaced with a V5 tag. For imaging, NAA80_FL and NAA80_ΔN were cloned between the BglII and HindIII sites of vector EGFP-C1 (Clontech Laboratories).

Rebowski G., Boczkowska M., Drazic A., Ree R., Goris M., Arnesen T, & Dominguez R. (2020). Mechanism of actin N-terminal acetylation. Science Advances, 6(15), eaay8793.

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Generation of Chimeric Immune Receptors

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Generation of CD16^158F-CD8α-CD28-CD3ζ CR has previously been described.^{16 (link)} The extracellular region of CD32A^131R was amplified by reverse-transcriptase polymerase chain reaction (RT-PCR) from RNA extracted from peripheral blood mononuclear cells (PBMCs) utilizing the following primers: forward 5′-GAGAATTCACCATGACTATGGAGACCCAAATG-3′ and reverse 5′-CGTACGCCCCATTGGTGAAGAGCTGCC-3′ (Thermo Fisher Scientific, Waltham, MA). PCR product was fused by restriction enzyme-compatible ends with the CD8α-CD28-CD3ζ domain contained in the pcDNA3.1/V5-His TOPO TA (Invitrogen, Carlsbad, CA). CD16^158F-CR and CD32^131R-CR were subcloned into NcoI and MluI sites of the SFG retroviral vector. CD16^158V and CD32A^131H were assembled by using synthetic oligonucleotides. Fragments were separately inserted into SFG vector and sequenced by GeneArt Gene Synthesis team (Invitrogen-Thermo Fisher Scientific, Regensburg, Germany).

Arriga R., Caratelli S., Lanzilli G., Ottaviani A., Cenciarelli C., Sconocchia T., Spagnoli G.C., Iezzi G., Roselli M., Lauro D., Coppola A., Dotti G., Ferrone S, & Sconocchia G. (2019). CD16-158-valine chimeric receptor T cells overcome the resistance of KRAS-mutated colorectal carcinoma cells to cetuximab. International journal of cancer, 146(9), 2531-2538.

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Porcine HSP70 Overexpression in IPEC-J2 Cells

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Porcine HSP70 encoding sequences were ligated into mammalian expression vector pcDNA3.1/V5-His-TOPO TA, procured from Invitrogen (Shanghai, China). The plasmid DNAs were extracted with an endotoxin-free Plasmid Maxi Kit (Qiagen, Valencia, CA, USA). IPEC-J2 cells were incubated in a 6-well tissue culture plate in culture medium (DMEM-F12: Gibco, Grand Island, NY, USA) supplemented with 100 mg/mL streptomycin, 100 U/mL penicillin, and 10% fetal bovine serum at a concentration of 1×10 6 cells/mL (5% CO 2 , 95% air, 100% humidity). IPEC-J2 cells were grown in plate at 85 95% con uence and then transfected with plasmid DNA encoding porcine HSP70 or empty vector as a control by Lipofectamine ® -3000 (Invitrogen, Shanghai, China).

Li J., Ju X., Yu T., Fang B., Wu L., Hu C., Liu X., Yu Z., Ma X., Gooneratne R., Spiteller M., & Ju X. (2020). Over-expression of HSP70 induces apoptosis of intestinal epithelial cells in heat-stressed pigs: A proteomic approach.

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CLDN10B Gene Cloning and Verification

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Human CLDN10B was cloned by PCR from a pool of cDNA from human renal biopsy specimens as previously described 17 using CLDN10B-specific primers: forward 5'-GC CGCCATGGCTAGCACG-3'and reverse 5'-GACATAAG-CATTTTTATCAAACTGTT TTGAAGGG-3'. PCR product was subcloned into pcDNA3.1/V5-HIS TOPO TA (Invitrogen, Villebon-sur-Yvette, France). PCR product insertion was verified by enzymatic digestions with EcoRI and BamHI. Construct was confirmed by sequencing (ATGC).

Hadj-Rabia S., Brideau G., Al-Sarraj Y., Maroun R.C., Figueres M.L., Leclerc-Mercier S., Olinger E., Baron S., Chaussain C., Nochy D., Taha R.Z., Knebelmann B., Joshi V., Curmi P.A., Kambouris M., Vargas-Poussou R., Bodemer C., Devuyst O., Houillier P, & El-Shanti H. (2018). Multiplex epithelium dysfunction due to CLDN10 mutation: the HELIX syndrome. Genetics in medicine : official journal of the American College of Medical Genetics, 20(2).

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