Pcr8 gw topo entry vector

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PCR8/GW/TOPO entry vector is a plasmid used for the cloning and expression of DNA sequences. It contains the necessary elements for efficient cloning and subsequent transfer of the insert into other expression vectors via the Gateway cloning system.

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Lab products found in correlation

Gateway lr clonase 2 enzyme mix, by Thermo Fisher Scientific (2 mentions) Foxa1 sirna, by Horizon Discovery (2 mentions) Control sirna luciferase gl2 duplex, by Horizon Discovery (2 mentions) Control and pgipz lentiviral shrnamir targeting, by Thermo Fisher Scientific (2 mentions) Plenti cmv to puro dest, by Addgene (2 mentions) Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions) Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions) Kpni enzyme, by Thermo Fisher Scientific (1 mentions) T4 dna ligase, by Thermo Fisher Scientific (1 mentions) Lr clonase enzyme mix, by Thermo Fisher Scientific (1 mentions) Ez c1 confocal microscope, by Nikon (1 mentions) Pcdna3.2 v5 dest vector, by Thermo Fisher Scientific (1 mentions) Anti mor antibody, by GeneTex (1 mentions) Platinum taq dna polymerase high fidelity enzyme, by Thermo Fisher Scientific (1 mentions) Herculase 2 fusion dna polymerase, by Agilent Technologies (1 mentions) Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions) Gateway lr clonase 2, by Thermo Fisher Scientific (1 mentions) Pfu turbo dna polymerase, by Agilent Technologies (1 mentions) Qiaprep spin miniprep kit, by Qiagen (1 mentions)

Close spelling variants of this product

PCR8/GW/TOPO Gateway entry vector PCR8/GW/TOPO vector PCR8/GW/TOPO PCR8 TOPO vector PCR8 vector TOPO vector

13 protocols using pcr8 gw topo entry vector

Construction of pMDC32-GUS control vectors

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To generate the pMDC32-GUS control construct; first, the GUS open reading frame was amplified by PCR using the Phusion high-fidelity DNA polymerase (Invitrogen, Carlsbad, CA, USA). Subsequently, GUS amplicon was introduced into the pCR8/GW/TOPO entry vector (Invitrogen) and then it was sub-cloned into the pMDC32 plant expression vector (Curtis & Grossniklaus, 2003 (link)) by attL/attR recombination sites using Gateway LR Clonase II Enzyme Mix (Invitrogen).
Each pMDC32-GUS::OpsDHN1 (full-length, PEST-1, PEST-2, and PEST-1-2) derived construct was generated by the fusion of two PCR products, the first one containing a version of the GUS open reading frame without a stop codon, and the second one with a stop codon, either OpsDHN1 open reading frame, OpsDHN1 nucleotides 372-594 (PEST-1), nucleotides 561-747 (PEST-2) or nucleotides 372-747 (PEST-1-2). To fuse the GUS and OpsDHN1 amplicons, the Kpn I recognition sequences were introduced in oligonucleotide sequence, and after PCR amplification these were digested with Kpn I enzyme (Invitrogen) generating cohesive ends and fused using T4 DNA ligase (Invitrogen). The ligated products were cloned into the pCR8/GW/TOPO entry vector and then sub-cloned into the pMDC32 vector as mentioned before. Selected clones were sequenced using the M13 forward primer.

Salazar-Retana A.L., Maruri-López I., Hernández-Sánchez I.E., Becerra-Flora A., Guerrero-González M.D, & Jiménez-Bremont J.F. (2019). PEST sequences from a cactus dehydrin regulate its proteolytic degradation. PeerJ, 7, e6810.

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Overexpression of AtPHT1;1 Variants

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The full-length open reading frame of WT AtPHT1;1 and its mutation variants were cloned into the pCR8/GW/TOPO entry vector (Invitrogen) and validated by sequencing. All variants were recombined into the destination vector of pMDC32 via LR Clonase enzyme mix (Invitrogen), in which the cauliflower mosaic virus (CaMV) 35S promoter (p35S) was replaced with AtPHT1;1 promoter (3319 bp containing 5′ UTR and the first intron). The WT and individual variant of AtPHT1;1 driven by its native promoter were introduced into the pht1;1 mutant by an Agrobacterium tumefaciens (strain GV3101) dipping method (Clough and Bent, 1998 (link)). Transgenic plants were selected by 20 μg/ml hygromycin. For each variant, 8-10 independent T2 lines were examined.

Liao Y.Y., Li J.L., Pan R.L, & Chiou T.J. (2019). Structure–Function Analysis Reveals Amino Acid Residues of Arabidopsis Phosphate Transporter AtPHT1;1 Crucial for Its Activity. Frontiers in Plant Science, 10, 1158.

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BiFC Analysis of RACK1A Interaction

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Plasmids for BiFC analysis were kindly provided by Dr. Stanton Gelvin of Purdue University. PCR-amplified coding regions of wild type RACK1A or the mutant Y248F-RACK1A were introduced into the pCR8/GW/TOPO entry vector (Invitrogen, Carlsbad, CA, USA). Entry clones were maintained in the Escherichia coli TOP10 strain, in LB medium supplemented with spectinomycin (50 μg/ml). Following manufacturer’s instruction for gateway cloning, the RACK1A coding sequences were cloned in the respective (pSAT5-DEST-cEYFP-C1(B) (pE3130)) and (pSAT4-DEST-nEYFP-C1 (pE3136) destination vectors). The destination vectors were maintained in DH5alpha E. coli strain on LB plates supplemented with 100 μmg/ml ampicillin. Transient gene expression in onion epidermal cells was performed using a High Efficiency Helios Gene Gun System (Bio-Rad) according to the published protocol (Hollender and Liu, 2010 (link)). After bombardment, the onion peels were incubated for 24 h on Murashige and Skoog plates in the dark. Images were obtained by using a Nikon EZ-C1 confocal microscope. The argon (488 nm) laser with appropriate emission filters was used for the visualization of FITC. The UV excitation (405 nm) was used to view the DAPI staining.

Sabila M., Kundu N., Smalls D, & Ullah H. (2016). Tyrosine Phosphorylation Based Homo-dimerization of Arabidopsis RACK1A Proteins Regulates Oxidative Stress Signaling Pathways in Yeast. Frontiers in Plant Science, 7, 176.

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Molecular Cloning and Manipulation of Androgen Receptor

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FOXA1 siRNA (sense 5′-GAGAGAAAAAAUCAACAGC-3′; antisense 5′-GCUGUUGAUUUUUUCUCUC-3′)^{14 (link),20 (link)}, AR on-target plus smart pool (L-003400-00-0020), and control siRNA Luciferase GL2 Duplex (D-001100-01-20) were synthesized by Dharmacon. The control and pGIPZ lentiviral shRNAmir targeting FOXA1 (Clone ID# V2LHS_16780) were purchased from Open Biosystems. C-terminal 3×HA-tagged full-length FOXA1 was cloned into a Tet-On inducible lentiviral vector that was kindly provided by Professor Junjie Chen (M.D. Anderson Cancer Center). The full-length (wild-type) and truncated AR (Q640X) were amplified from pcDNA3.1-AR^fl plasmid^{24 (link)} and cloned into the pCR8/GW/TOPO entry vector (Invitrogen). The pCR8-AR C562S and G568W mutants were generated from pCR8-AR wildtype using QuikChange® II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Lentiviral constructs were generated by LR recombination between pCR8-AR constructs and pLenti CMV/TO Puro DEST (Addgene plasmid 17293). Details regarding cloning primers and plasmid construction were provided in Supplementary Table 1 and Supplementary Table 2.

Jin H.J., Zhao J.C., Wu L., Kim J, & Yu J. (2014). Cooperativity and Equilibrium with FOXA1 Define the Androgen Receptor Transcriptional Program. Nature communications, 5, 3972.

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Molecular Cloning and Manipulation of Androgen Receptor

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Jin H.J., Zhao J.C., Wu L., Kim J, & Yu J. (2014). Cooperativity and Equilibrium with FOXA1 Define the Androgen Receptor Transcriptional Program. Nature communications, 5, 3972.

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Overexpression of BrLAS in Arabidopsis

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To construct the overexpression vector 35S: BrLAS, the entire 1,332-bp coding sequence of BrLAS was amplified and subcloned into the pCR8/GW/TOPO entry vector (Invitrogen) according to the manufacturer’s instructions. The CDS fragment was then inserted into the pMDC32 Gateway-compatible binary vector through LR recombination reactions (Curtis and Grossniklaus, 2003 (link)). The 35S: BrLAS plasmid was then introduced into Agrobacterium tumefaciens strain GV3101 and transformed into Arabidopsis Col-0 plants using the floral dip method (Clough and Bent, 1998 (link)).

Li P., Zhang B., Su T., Li P., Xin X., Wang W., Zhao X., Yu Y., Zhang D., Yu S, & Zhang F. (2018). BrLAS, a GRAS Transcription Factor From Brassica rapa, Is Involved in Drought Stress Tolerance in Transgenic Arabidopsis. Frontiers in Plant Science, 9, 1792.

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Cloning and Expression of MOR1-Myc Fusion Protein

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Myc-DDK-tagged ORF clone of Homo sapiens opioid receptor, mu 1 (OPRM1), transcript variant MOR-1 (OriGene Technologies Inc, MD) was amplified using Platinum Taq DNA polymerase high fidelity enzyme (Invitrogen, CA) and subsequently cloned into a pCR8/GW/Topo entry vector (Invitrogen, CA) according to manufacturer's instructions. Plasmid DNA was extracted from selected clones by QIAquick Plasmid Mini kit (Qiagen, CA). ORF integrity and fragment orientation were confirmed by sequencing. The MOR1-Myc fusion product was then transferred to pcDNA3.2/v5 DEST vector (Invitrogen, CA) by LR reaction. The resulting construct (pcDNA3.2-MOR1-Myc) was transfected into H358 cells using FuGENE HD™ as the transfection reagent (Roche Applied Sciences) according to the protocol provided by Roche as we have previously described. Cells (∼40% confluent) were serum-starved for 1 hour followed by incubation with pcDNA3.2-MOR1-Myc for 6 hours in serum-free media. Serum-containing media was then added (10% serum final concentration) for 42 hours and neomycin selection reagent was added. Overexpression was confirmed by immunoblot analysis with anti-MOR antibody (GeneTex, San Antonio, TX).

Lennon F.E., Mirzapoiazova T., Mambetsariev B., Poroyko V.A., Salgia R., Moss J, & Singleton P.A. (2014). The Mu Opioid Receptor Promotes Opioid and Growth Factor-Induced Proliferation, Migration and Epithelial Mesenchymal Transition (EMT) in Human Lung Cancer. PLoS ONE, 9(3), e91577.

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Lentiviral Expression of Mutant FANCA

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Mutant FANCA expression plasmids were obtained by site-directed mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit, Agilent) from a wild-type open reading frame (ORF) construct (the primers used for generating FANCA mutations are listed in Table 2). Mutant and wild-type FANCA ORFs were then polymerase chain reaction (PCR)-amplified using high-fidelity Taq polymerase (Herculase II fusion DNA polymerases, Agilent) and primers that generated a FANCA ORF with a carboxy-terminal Flag tag. PCR fragments were cloned into pcr8/gw/topo entry vector (Invitrogen) and Gateway-cloned into a lentiviral expression vector (pHAGE_CMV_IP_HAFLAG; kindly provided by A. Smogorzewska) and Sanger sequence–verified. Each expression plasmid was transfected together with the packaging plasmids (SV40 Gag/Pol, SV40 VSVG, SV40 Rev, and SV40 Tat) described previously (Mostoslavsky et al. 2005 (link)). Supernatant containing lentiviral particles were collected, filtered, and placed on RA3087 cells with polybrene at a final concentration of 8 µg/ml. Following 24 h incubation, the media containing the virus is removed, and fresh complete media is added. Seventy-two hours after infection, the media is replaced with fresh media containing puromycin at 1 µg/ml. Following 7 d of puromycin selection, cells were collected to check for FANCA protein expression.

Wilkes D.C., Sailer V., Xue H., Cheng H., Collins C.C., Gleave M., Wang Y., Demichelis F., Beltran H., Rubin M.A, & Rickman D.S. (2017). A germline FANCA alteration that is associated with increased sensitivity to DNA damaging agents. Cold Spring Harbor Molecular Case Studies, 3(5), a001487.

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Tomato ABA Receptor-Phosphatase Interactions

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The full-length coding sequences of Sl08g076960, Sl06g061180, Sl09g015380, Sl06g050500, Sl03g095780, Sl12g055990, and Sl03g007310 ABA receptors as well as Sl05g052980 PP2C were amplified by PCR from tomato leaf/fruit cDNA and cloned into the pCR8/GW/TOPO entry vector (Invitrogen). An N-terminal deleted version (ΔN) of Sl12g096020 PP2C was amplified from tomato leaf/fruit cDNA using primers that amplify the catalytic PP2C core (amino acid residues 178–509, ΔN Sl12g096020). All the primers used in this work are listed in Supplementary Table S1 available at JXB online. Appropriate restriction sites were introduced in some primers to allow the subsequent cloning steps, and all constructs were verified by DNA sequencing. Tomato ABA receptors were fused by Gateway recombination to the GAL4 DNA-binding domain (GBD) in pGBKT7GW. As preys, a set of Arabidopsis clade A PP2Cs fused to the GAL4 activation domain (GAD) in the pGADT7 vector was used (Lackman et al., 2011 ; Antoni et al., 2012 (link)). Tomato Sl05g052980 and ΔN Sl12g096020 PP2Cs were fused to the GAD in the pGADT7GW vector. Protocols for Y2H assays were similar to those described previously (Saez et al., 2008 (link)).

González-Guzmán M., Rodríguez L., Lorenzo-Orts L., Pons C., Sarrión-Perdigones A., Fernández M.A., Peirats-Llobet M., Forment J., Moreno-Alvero M., Cutler S.R., Albert A., Granell A, & Rodríguez P.L. (2014). Tomato PYR/PYL/RCAR abscisic acid receptors show high expression in root, differential sensitivity to the abscisic acid agonist quinabactin, and the capability to enhance plant drought resistance. Journal of Experimental Botany, 65(15), 4451-4464.

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Characterization of the AtGRDP2 Promoter

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The AtGRDP2 promoter region (2 kb upstream of the start codon) was PCR-amplified from the Arabidopsis genomic DNA. Amplification was carried out with primers ATPROM37fw and ATPROM37rv (Table S1). The fragment was cloned into pCR®8/GW/TOPO® entry vector (Invitrogen) and fused by recombination to the GUS-GFP reporter genes in the pKGWFS7 binary vector (Karimi et al., 2002 (link)). Agrobacterium tumefaciens GV2260 strain harboring the AtGRDP2 promoter::GUS-GFP construction was used for Arabidopsis transformation, as described before. Five independent transgenic lines were selected on 50 μg/mL kanamycin. T3 homozygous seedlings were used for GUS histochemical analysis as described below.

Ortega-Amaro M.A., Rodríguez-Hernández A.A., Rodríguez-Kessler M., Hernández-Lucero E., Rosales-Mendoza S., Ibáñez-Salazar A., Delgado-Sánchez P, & Jiménez-Bremont J.F. (2015). Overexpression of AtGRDP2, a novel glycine-rich domain protein, accelerates plant growth and improves stress tolerance. Frontiers in Plant Science, 5, 782.

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