Pcdna6.2 v5 dest

Manufactured by Thermo Fisher Scientific

Sourced in United States

PcDNA6.2/V5-DEST is a mammalian expression vector that allows for the expression of recombinant proteins with a C-terminal V5 epitope tag. The vector features a CMV promoter for high-level expression in a variety of cell lines and a blasticidin resistance gene for selection of stable cell lines.

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8 protocols using pcdna6.2 v5 dest

Molecular Cloning and Plasmid Construction

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MALT1A and mutants thereof were cloned into pCMVSport, encoding an N-terminal FLAG-His-Strep tag. BCL10 and CARD11-L244P were cloned into pcDNA6.2/V5-DEST (Invitrogen). The TRAF6-expressing plasmid was described previously [21 (link)] as well as the plasmid encoding A20 [22 (link)]. The plasmids for expression of CYLD, MALT1 isoform B and TRAF2 were obtained from GeneCopoeia™ (pReceiver-M11 vector) as well as the plasmid encoding TRAF3 (pReceiver-M12 vector). The TRAF6-binding deficient MALT1 construct (MALT1-E4/A, N-ter. FLAG-tagged) was obtained from the BCCM/LMBP plasmid collection. Oligonucleotides for mutagenesis reactions are described elsewhere (S1 Table).

Ginster S., Bardet M., Unterreiner A., Malinverni C., Renner F., Lam S., Freuler F., Gerrits B., Voshol J., Calzascia T., Régnier C.H., Renatus M., Nikolay R., Israël L, & Bornancin F. (2017). Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation. PLoS ONE, 12(1), e0169026.

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Mouse PPARGC1A cDNA Cloning and Expression

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The cDNA for mouse PPARGC1A (NM_008904.2) was cloned from a brown fat cDNA library and inserted into pDONR221 using BP clonase (Invitrogen). Site-directed mutagenesis was performed using QuikChange IIXL (Stratagene). For expression, plasmids were then cloned into pcDNA6.2 V5 dest (Invitrogen) using LR reaction.

Dumesic P.A., Egan D.F., Gut P., Tran M.T., Parisi A., Chatterjee N., Jedrychowski M., Paschini M., Kazak L., Wilensky S.E., Dou F., Bogoslavski D., Cartier J.A., Perrimon N., Kajimura S., Parikh S.M, & Spiegelman B.M. (2019). An evolutionarily conserved uORF regulates PGC1α and oxidative metabolism in mice, flies, and bluefin tuna. Cell metabolism, 30(1), 190-200.e6.

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Cloning and Mutation of Human GARS

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The cytoplasmic isoform of human GARS was cloned into the pcDNA6.2/C-EmGFP-DEST and pcDNA6.2/V5-DEST vectors (Invitrogen) to express GlyRS with a C-terminal eGFP or V5 tag, respectively. Smn with a C-terminal eGFP tag was expressed from pEGFP-N1 (43 (link)). To create the GARS^P234KY and ΔWHEP (residues 71–126) mutation constructs, a Q5 Site-Directed Mutagenesis Kit (New England Biolabs) was used, as per the manufacturer's instructions, with the following primers: WT-P234KY_F 5′-TTT CAT TGG GAA ATA TGG AGG AAA CAT GC-3′, WT-P234KY_R 5′-GTC TTG AAC ATT AAG TTA AAA GAC-3′, ddWHEP_F 5′-ACC CTG AAG AGG AGG TTT TTC-3′ and ddWHEP_R 5′-TGC TAG CCT CAG AGG TGC-3′.

Grice S.J., Sleigh J.N., Motley W.W., Liu J.L., Burgess R.W., Talbot K, & Cader M.Z. (2015). Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology. Human Molecular Genetics, 24(15), 4397-4406.

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HEK293 Cell Transfection with OsKAT2 Variants

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HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM with 4,500 mg/l glucose; Gibco, USA) containing 2 mM glutamine, 100 U/ml penicillin/streptomycin and 10% FBS (Invitrogen, USA). The cells were transfected with 6 μg of mammalian expression vector pcDNA6.2 V5-DEST (Invitrogen, USA) containing wild-type OsKAT2, OsKAT2 T235R, OsKAT2 T285A, and OsKAT2 T285D according to the manufacturer’s protocol using Lipofectamine 2000 (Invitrogen, USA). Transfected cells were incubated in DMEM medium and maintained at 37°C in a humidified incubator in the presence of 5% CO₂ (Sanyo, Japan). For electrophysiological studies, cells were spread on cover slips coated with 0.1% poly-D-lysine (Sigma-Aldrich, USA). The experiments were performed using cells incubated for 1–2 days after transfection.

Moon S.J., Kim H.Y., Hwang H., Kim J.A., Lee Y., Min M.K., Yoon I.S., Kwon T.R, & Kim B.G. (2017). A Dominant Negative OsKAT2 Mutant Delays Light-Induced Stomatal Opening and Improves Drought Tolerance without Yield Penalty in Rice. Frontiers in Plant Science, 8, 772.

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GM-CSF Receptor Subunits Interaction

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The complete coding regions of CSF2RA, CSF2RB_WT and CSF2RB_Mut were synthesized by GeneArt gene synthesis and cloned into the Gateway pDONR™221 Vector (GeneArt, Life Technologies), and the clone sequences confirmed by Sanger sequencing. For immunoblot analyses, CSF2RB_WT and Mut were sub-cloned into the Gateway destination vector pcDNA6.2_C-EmGFP-DEST (Life-Technologies), and CSF2RA into pcDNA6.2_C-YFP-DEST (Life-Technologies). For co-localization studies, CSF2RA and CSF2RB_WT were sub-cloned into PCDNA6.2/V5-DEST (Life-technologies) and pDEST-mcherry-N1 (Addgene plasmid # 31907), respectively.
HEK293 cells (ATCC CRL 1573) were seeded at 2×10⁵ cells per well in 24 well plates and transfected with 500ng of total DNA with Lipofectamine 3000 (Life Technologies). Transfected DNA was comprised of combinations of pcDNA6.2_C-YFP-DEST -CSF2RA, pcDNA6.2_C-EmGFP-DEST-CSF2RB_WT, and pcDNA6.2_C-EmGFP-DEST-CSF2RB_Mut (Life Technologies). Twenty-four hours post-transfection, cells were stimulated with 0, 0.4, 2 or 10 ng/ml of GM-CSF for 15 min and lysed in RIPA buffer (Millipore). For co-localization studies, CSF2RA, CSF2RB_WT and CSF2RB_Mut were co-transfected and stimulated with GM-CSF (10 ng/ml).

Chuang L.S., Villaverde N., Hui K.Y., Mortha A., Rahman A., Levine A.P., Haritunians T., Evelyn Ng S.M., Zhang W., Hsu N.Y., Facey J.A., Luong T., Fernandez-Hernandez H., Li D., Rivas M., Schiff E.R., Gusev A., Schumm L.P., Bowen B.M., Sharma Y., Ning K., Remark R., Gnjatic S., Legnani P., George J., Sands B.E., Stempak J.M., Datta L.W., Lipka S., Katz S., Cheifetz A.S., Barzilai N., Pontikos N., Abraham C., Dubinsky M.J., Targan S., Taylor K., Rotter J.I., Scherl E.J., Desnick R.J., Abreu M.T., Zhao H., Atzmon G., Pe’er I., Kugathasan S., Hakonarson H., McCauley J.L., Lencz T., Darvasi A., Plagnol V., Silverberg M.S., Muise A.M., Brant S.R., Daly M.J., Segal A.W., Duerr R.H., Merad M., McGovern D.P., Peter I, & Cho J.H. (2016). A Frameshift in CSF2RB Predominant Among Ashkenazi Jews Increases Risk for Crohn’s Disease and Reduces Monocyte Signaling via GMCSF. Gastroenterology, 151(4), 710-723.e2.

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Zinc Homeostasis in Mesenchymal Stem Cells

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Cell culture and materials hMSCs were purchased (Lonza, Basel, Switzerland) and maintained in DMEM medium (Gibco, Carlsbad, CA) with 10% fetal bovine serum and antibiotics, at 37 C. siRNA transfection was performed with ON-TARGETplus SMARTpool siRNA (Life Technologies, Carlsbad, CA) using Lipofectamine RNAimax (Life Technologies) according to the manufacturer's instructions. Fluozin-3 (Life Technologies) and zinc pyrithione (Sigma, St. Louis, MO) were dissolved in DMSO and stocked at À80 C before use. Fibrogenic differentiation was induced by the connective-tissue growth factor treatment (Lee et al., 2010) . Osteogenic differentiation was induced by a StemPro Osteoblast Differentiation Kit (Gibco). Masson Trichrome (Sigma), Alizarin red (Sciencell Research Laboratories, Carlsbad, CA), and Safranin O (Sigma) were used to stain differentiated cells. To construct V5-tagged ZIP7, mouse cDNA was ligated into pcDNA6.2/V5-DEST (Life Technologies) as previously described (Bin et al., 2011) .

Bin B.H., Bhin J., Seo J., Kim S.Y., Lee E., Park K., Choi D.H., Takagishi T., Hara T., Hwang D., Koseki H., Asada Y., Shimoda S., Mishima K, & Fukada T. (2017). Requirement of Zinc Transporter SLC39A7/ZIP7 for Dermal Development to Fine-Tune Endoplasmic Reticulum Function by Regulating Protein Disulfide Isomerase. The Journal of investigative dermatology, 137(8).

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Generation of Tau-Specific Antibody RNJ1

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RNJ1 is a tau-specific antibody that was isolated following the panning of the Tomlinson I and J scFv library with recombinant full-length hTau. To generate the RNJ1 IgG, the variable heavy and variable light sequences were cloned into murine IgG1 and kappa mAbXpress vectors.^{22 (link)} Large-scale low-endotoxin recombinant antibody production was conducted (Queensland node of the National Biologics Facility) and the final preparation was stored in 1× phosphate-buffered saline at −80°C. The hTau-specific antibody, HJ8.5, was a gift from Professor Jürgen Götz at the University of Queensland and was generated as previously described.^{1 (link)} A mammalian expression plasmid encoding the RNJ1 scFv was generated by cloning the variable light and variable heavy domains, joined by a (Gly4Ser)3 linker and in frame with a C-terminal triple Flag tag, into pcDNA™6.2/V5-DEST (Thermo Fisher Scientific).

Wongsodirdjo P., Caruso A.C., Yong A.K., Lester M.A., Vella L.J., Hung Y.H, & Nisbet R.M. (2024). Messenger RNA-encoded antibody approach for targeting extracellular and intracellular tau. Brain Communications, 6(2), fcae100.

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Cloning and Mutagenesis of HCN Ion Channel Constructs

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The plasmid (pENTR223.1-HCN4) containing the human HCN4 cDNA (HsCD00350637) was ordered from DNASU Plasmid Repository (http://dnasu.org/DNASU/GetCloneDetail.do?cloneid=350637). The HCN4 cDNA was cloned into the CVM promoter containing expression vector pcDNA6.2-V5-DEST by Gateway cloning (ThermoFisher Scientific). Human HCN1 (NM_021072.4) and HCN2 (NM_001194.4) cDNAs were synthesized by TWIST Bioscience and cloned into the pTwist-CMV-BetaGlobin-WPRE-Neo mammalian expression vector. Single and double mutations targeting the candidate residues identified by computational modeling were generated by the Q5 Site-directed mutagenesis kit (New England BioLabs). More complex compound mutations were generated in synthetic gene fragments (TWIST Bioscience), which were then used to replace the corresponding regions in the wildtype plasmids with NEBuilder^® HiFi DNA Assembly Cloning Kit (New England BioLabs). All plasmids were fully sequenced. The rabbit M408Q/Y410F (corresponding to M407Q/Y409F on mouse and human HCN4) were generated by the Q5 Site-directed mutagenesis kit.

Wu Y., Wang Q., Granger J., Gaido O.R., Aguilar E.N., Ludwig A., Moroni A., Bianchet M.A, & Anderson M.E. (2023). HCN channels sense temperature and determine heart rate responses to heat. bioRxiv.

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