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Gateway lr recombination reaction

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The Gateway LR recombination reaction is a laboratory technique used to transfer DNA sequences from an entry clone to an expression vector. It facilitates the cloning of DNA fragments into different vectors for downstream applications.

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Lab products found in correlation

Pentr d topo vector, by Thermo Fisher Scientific (3 mentions) Pdonr207 vector, by Thermo Fisher Scientific (3 mentions) Gateway bp recombination reaction, by Thermo Fisher Scientific (3 mentions) Proquest two hybrid system, by Thermo Fisher Scientific (2 mentions) Quikchange lighting multi site directed mutagenesis kit, by Agilent Technologies (2 mentions) Gateway bp reaction, by Thermo Fisher Scientific (2 mentions) Platinum taq dna polymerase high fidelity kit, by Thermo Fisher Scientific (2 mentions) Anti ha antibody 3f10, by Roche (2 mentions) Gateway compatible modified ptnt vectors, by Promega (2 mentions) Tnt sp6 high yield wheat germ protein expression system, by Promega (2 mentions) Pcr8 gw topo ta cloning kit, by Thermo Fisher Scientific (1 mentions) Kod plus dna polymerase, by Toyobo (1 mentions) Gateway recombination cloning system, by Thermo Fisher Scientific (1 mentions) Pdonrzeo vector, by Thermo Fisher Scientific (1 mentions) Pentr d topo plasmid, by Thermo Fisher Scientific (1 mentions) Pentr2b, by Thermo Fisher Scientific (1 mentions) Geneart, by Thermo Fisher Scientific (1 mentions) Phosphatase inhibitors 1 2, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Micropulser electroporation system, by Bio-Rad (1 mentions)

25 protocols using gateway lr recombination reaction

1

Yeast Two-Hybrid Analysis of GI Interactions

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The ProQuest™ Two-Hybrid System (Invitrogen) was used to perform yeast two-hybrid (Y2H) analyses. The cDNA encoding full-length GI was transferred from the pENTR/D-TOPO vector (Invitrogen) into the pDEST32 vector by Gateway LR recombination reaction (Invitrogen) to generate the bait plasmid. The pDEST22 prey plasmids containing the sequences encoding PIF1, PIF3, PIF4, and PIF5 have been previously described (Pruneda-Paz et al., 2014 (link)). Empty pDEST22 and the pExpAD502 plasmids were used as negative controls. All Y2H procedures were performed following the manufacturer’s instructions. Quantitation of β-galactosidase activity was performed in a 96-well format as previously described (Pruneda-Paz et al., 2009 (link)).
Nohales M.A., Liu W., Duffy T., Nozue K., Sawa M., Pruneda-Paz J.L., Maloof J.N., Jacobsen S.E, & Kay S.A. (2019). Multilevel modulation of light signaling by GIGANTEA regulates both the output and pace of the circadian clock. Developmental cell, 49(6), 840-851.e8.
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2

Arabidopsis Circadian Rhythm Reporter Lines

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The TOC1p:LUC (Col-0), LHYp:LUC (Col-0)14 and CAB2p:LUC (Col-0) seeds were provided by Dr. Robertson McClung and the toc1-101 mutant31 (link) by Dr. Shu-Hsing Wu. Mutants of npr1-318 (link), sid216 (link), trx-h319 (link) and trx-h519 (link) were used to cross with the luciferase reporter lines. 35S:NPR1-GFP (in npr1-1)12 (link) plants were used in ChIP experiment. To generate CAT3p:LUC, CAT2p:LUC homozygous lines and different T1 lines of TOC1p:LUC and TOC1p(TBSm):LUC (TOC1 promoter with mutated TGA-binding sites), wild-type CAT3, CAT2 and TOC1 promoters and mutated TOC1 promoter (amplified using QuikChange Lighting Multi Site-directed mutagenesis kit, Agilent Technologies) were cloned into the pDONR207 vector (Invitrogen) through the Gateway BP reaction (Invitrogen) and then transferred to the destination vector pGWB23532 (link) through the Gateway LR recombination reaction (Invitrogen). Agrobacterium-mediated transformation of Arabidopsis was performed as previously described using wild-type plants33 (link). Homozygous T3 lines of CAT2p:LUC, CAT3p:LUC and different T1 lines of TOC1p:LUC and TOC1p(TBSm):LUC were selected and used for the luciferase imaging experiment. All primer sequences used for making the transgenic constructs are listed in Extended Data Table 1.
Zhou M., Wang W., Karapetyan S., Mwimba M., Marqués J., Buchler N.E, & Dong X. (2015). Redox rhythm reinforces the circadian clock to gate immune response. Nature, 523(7561), 472-476.
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3

HvCCA1 Overexpression in Arabidopsis

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The entire HvCCA1 transcript was amplified using KOD TAQ polymerase (Novagen). HvCCA1 was cloned into GATEWAY pCR8/GW/TOPO entry vector using pCR8/GW/TOPO TA cloning kit (Invitrogen) following the manufacturer’s instructions. HvCCA1 was then subcloned into the binary plazmid PMDC32 using the Gateway LR recombination reaction (Invitrogen). The binary plasmid PMDC32 confers hygromycin resistance in plants and contains a double 35S cauliflower mosaic virus promoter for constitutive expression of the inserted gene [16 (link)]. The CaMV35S:HvCCA1 was introduced into GV3101 Agrobacterium tumefaciens and subsequently into Arabidopsis thaliana Ws-2 via the floral dip method [17 (link)]. Out of 21 primary transformants, nine were identified as homozygous lines carrying an insert at a single locus. HvCCA1 overexpression was confirmed by RT-PCR (S1 Fig). Homozygous lines were used for all experiments.
Kusakina J., Rutterford Z., Cotter S., Martí M.C., Laurie D.A., Greenland A.J., Hall A, & Webb A.A. (2015). Barley Hv CIRCADIAN CLOCK ASSOCIATED 1 and Hv PHOTOPERIOD H1 Are Circadian Regulators That Can Affect Circadian Rhythms in Arabidopsis. PLoS ONE, 10(6), e0127449.
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4

Yeast Two-Hybrid Assay for GI Interactions

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We used the ProQuest Two-Hybrid System (Invitrogen) as previously described (28 (link)). Specifically, the cDNA encoding full-length GI was transferred from the pENTR/D-TOPO vector (Invitrogen) into the pDEST32 vector by Gateway LR recombination reaction (Invitrogen) to generate the bait plasmid; the pDEST22 prey plasmids containing the sequences encoding RGA, GAI, RGL1, RGL2, and RGL3 have been previously described (45 (link)). Empty pDEST22 and the pExpAD502 plasmids were used as negative controls. Quantitation of β-galactosidase activity was performed in a 96-well format as previously described (47 (link)).
Nohales M.A, & Kay S.A. (2019). GIGANTEA gates gibberellin signaling through stabilization of the DELLA proteins in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 116(43), 21893-21899.
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5

Gateway Cloning of LEA5-YFP

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PCR products containing LEA5 joined in frame to YFP were cloned into pDONR207 using the Gateway BP recombination reaction (Invitrogen). After verification of the nucleotide sequence of the fragment, Gateway LR recombination reaction (Invitrogen) was used to transfer the LEA5-YFP into pBRACT214 vector containing ubiquitin promoter and hygromycin B resistance for positive selection of transformed plants.
Karpinska B., Razak N., Shaw D.S., Plumb W., Van De Slijke E., Stephens J., De Jaeger G., Murcha M.W, & Foyer C.H. (2022). Late Embryogenesis Abundant (LEA)5 Regulates Translation in Mitochondria and Chloroplasts to Enhance Growth and Stress Tolerance. Frontiers in Plant Science, 13, 875799.
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6

Comparative analysis of ZRS enhancer

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Orthologous ZRS sequences from various species where retrieved from the UCSC genome database (http://genome.ucsc.edu), the gar genome from the ENSEMBL database. and the skate genome from Skatebase. Sequence alignments and conservation peaks were visualized using the mVista program Shuffle-LAGAN. Putative ZRS elements were isolated from: mouse (Mus musculus), anole (Anolis carolinensis), coelacanth (Latimeria menadoensis), zebrafish (Danio rerio), gar (Lepisosteus oculatus), skate (Leucoraja erinacea), and sea lamprey (Petromyzon marinus). Using mouse or zebrafish as the reference genome, two Lmbr1-like genes were identified in the sea lamprey genome and the intronic region spanning exons five and six was subsequently tested for regulatory activity in transgenic assays. Supplementary Table 1 lists the oligonucleotide sequences used to amplify the genomic fragments from their corresponding genomes. Genomic DNA fragments were isolated using the Platinum® Taq DNA polymerase High Fidelity Kit (Life Technologies). Fragments were cloned into an entry vector (PCR8/GW/TOPO; except for D. rerio, which was cloned into pENTR/D-TOPO) and transferred to the appropriate destination vectors using the Gateway® LR recombination reaction (Invitrogen).
Letelier J., de la Calle-Mustienes E., Pieretti J., Naranjo S., Maeso I., Nakamura T., Pascual-Anaya J., Shubin N., Schneider I., Martinez-Morales J.R, & Gómez-Skarmeta J.L. (2018). A conserved Shh cis-regulatory module highlights a common developmental origin of unpaired and paired fins. Nature genetics, 50(4), 504-509.
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7

Cloning and Transformation of AaMYB17 in A. annua

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The full-length cDNA sequence of AaMYB17 was amplified using the cDNA of the A. annua meristem through PCR using KOD plus DNA polymerase (Toyobo, Osaka, Japan). It was further cloned into a pHB vector under a double CaMV35S promoter to generate pHB-CaMV35S:AaMYB17-YFP:NOS with the YFP fused to the C-terminal of AaMYB17 (Mao et al., 2005 (link)). A 269 bp AaMYB17 fragment was recombined into the phellsgate12 vector via a gateway LR recombination reaction (Invitrogen) to construct the AaMYB17-RNAi vector. A 2185 bp AaMYB17 promoter fragment was cloned for the construction of pCAMBIA 1391Z-PMYB17. These constructs were introduced into Agrobacterium tumefaciens strain EHA105, following Agrobacterium-mediated transformation of A. annua as described previously (Shen et al., 2016 (link)).
Qin W., Xie L., Li Y., Liu H., Fu X., Chen T., Hassani D., Li L., Sun X, & Tang K. (2021). An R2R3-MYB Transcription Factor Positively Regulates the Glandular Secretory Trichome Initiation in Artemisia annua L.. Frontiers in Plant Science, 12, 657156.
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8

Gateway Cloning of CaMKII Isoforms

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To construct expression vectors, we routinely used the Gateway recombination cloning system (Invitrogen). The BP clones encoding human CaMKIIα (NM_015981.3, Addgene, Watertown, Massachusetts, USA, 23408), human CaMKIIβ (NM_172080.2, Addgene, 23820), human CaMKIIδ (NM_172127.2, Addgene, 23814), and human CaMKIIγ (NM_172170.5, Addgene, 23409) were purchased from Addgene. These genes were transferred from BP clones to several destination expression vectors, such as pDEST-HA-N, pDEST-Flag-N, and pDEST-mCherry-N by the Gateway LR recombination reaction (Invitrogen), according to the manufacturer’s guidelines.
Sim K.M., Lee Y.S., Kim H.J., Cho C.H., Yi G.S., Park M.J., Hwang E.M, & Park J.Y. (2020). Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression. Cells, 9(5), 1079.
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9

In vitro protein interaction assay

Cited 3 times
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For in vitro pull-down assays, additional constructs were made. The pENTR/D-TOPO plasmids (Invitrogen) containing the sequences encoding GI, PIF1, and PIF5 have been previously described (Pruneda-Paz et al., 2014 (link); Sawa and Kay, 2011 (link)). Full-length PIF3, PIF4, and GFP sequences were amplified by PCR (primers used are listed in supplemental Table S2) and cloned into the pENTR/D-TOPO vector (Invitrogen). Partial PIF3 sequences were amplified by PCR (primers used are listed in supplemental Table S2) and cloned into the pDONRZeo vector (Invitrogen) by Gateway BP recombination reaction (Invitrogen). To express proteins in the cell-free system, all inserts were transferred by Gateway LR recombination reaction (Invitrogen) into Gateway compatible modified pTnT vectors (Promega) (Nito et al., 2013 (link)), which were kindly provided by Dr. Joanne Chory (The SALK Institute, La Jolla, CA). The vectors contained an N-terminal HA or Flag tag as specified in each case. Proteins were co-expressed using TnT® SP6 High-Yield Wheat Germ Protein Expression System (Promega) as per manufacturer’s instructions. Five percent of the reactions (2.5 μl) were used to verify expression of the proteins (input) and the remaining extract was immunoprecipitated as earlier described (Pedmale et al., 2016 (link)) using anti-HA 3F10 antibody (Roche).
Nohales M.A., Liu W., Duffy T., Nozue K., Sawa M., Pruneda-Paz J.L., Maloof J.N., Jacobsen S.E, & Kay S.A. (2019). Multilevel modulation of light signaling by GIGANTEA regulates both the output and pace of the circadian clock. Developmental cell, 49(6), 840-851.e8.
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10

Comparative analysis of ZRS enhancer

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Orthologous ZRS sequences from various species where retrieved from the UCSC genome database (http://genome.ucsc.edu), the gar genome from the ENSEMBL database. and the skate genome from Skatebase. Sequence alignments and conservation peaks were visualized using the mVista program Shuffle-LAGAN. Putative ZRS elements were isolated from: mouse (Mus musculus), anole (Anolis carolinensis), coelacanth (Latimeria menadoensis), zebrafish (Danio rerio), gar (Lepisosteus oculatus), skate (Leucoraja erinacea), and sea lamprey (Petromyzon marinus). Using mouse or zebrafish as the reference genome, two Lmbr1-like genes were identified in the sea lamprey genome and the intronic region spanning exons five and six was subsequently tested for regulatory activity in transgenic assays. Supplementary Table 1 lists the oligonucleotide sequences used to amplify the genomic fragments from their corresponding genomes. Genomic DNA fragments were isolated using the Platinum® Taq DNA polymerase High Fidelity Kit (Life Technologies). Fragments were cloned into an entry vector (PCR8/GW/TOPO; except for D. rerio, which was cloned into pENTR/D-TOPO) and transferred to the appropriate destination vectors using the Gateway® LR recombination reaction (Invitrogen).
Letelier J., de la Calle-Mustienes E., Pieretti J., Naranjo S., Maeso I., Nakamura T., Pascual-Anaya J., Shubin N., Schneider I., Martinez-Morales J.R, & Gómez-Skarmeta J.L. (2018). A conserved Shh cis-regulatory module highlights a common developmental origin of unpaired and paired fins. Nature genetics, 50(4), 504-509.
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