A putative Ern1 promoter region of 2,178 bp and a fulllength Ern1 in L. japonicus and a Rap2.11 in Arabidopsis genes were amplified from L. japonicus Gifu or A. thaliana Col-0 genomic DNA by PCR, using primers containing attB sites and recombined into pDONR207 (Invitrogen), using the Gateway BP reaction (Invitrogen) to create the entry clones pDONR207: pErn1, pDONR207:Ern1, and pDONR207:Rap2.11. Entry clones were recombined using Gateway LR reactions (Invitrogen) with destination vector pIV10:GW:GUS and pCAMBIA1300: Ljubiquitin promoter:GW (Maekawa et al. 2008) to create the pIV10:pErn1:GUS, pCAMBIA1300:Ljubiquitin promoter:Ern1, and pCAMBIA1300:Ljubiquitin promoter:Rap2.11 constructs.
Pdonr207
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The PDONR207 is a laboratory instrument designed for multi-parameter analysis. It features advanced optical detection and data processing capabilities to support various analytical applications. The core function of this product is to provide precise and reliable measurements, however, a detailed description of its intended use would require further information that is not available.
Automatically generated - may contain errors
Lab products found in correlation
Gateway lr reaction, by Thermo Fisher Scientific (6 mentions)
Bp reaction, by Thermo Fisher Scientific (6 mentions)
Gateway technology, by Thermo Fisher Scientific (5 mentions)
Lr reaction, by Thermo Fisher Scientific (5 mentions)
Pdonr201, by Thermo Fisher Scientific (5 mentions)
Gateway bp reaction, by Thermo Fisher Scientific (4 mentions)
Ah109, by Takara Bio (3 mentions)
Gateway cloning system, by Thermo Fisher Scientific (3 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (3 mentions)
Bp clonase 2 enzyme mix, by Thermo Fisher Scientific (3 mentions)
Lr clonase, by Thermo Fisher Scientific (3 mentions)
Pdest32, by Thermo Fisher Scientific (2 mentions)
Lr clonase 2, by Thermo Fisher Scientific (2 mentions)
Pearleygate202, by Thermo Fisher Scientific (2 mentions)
Geneart site directed mutagenesis system, by Thermo Fisher Scientific (2 mentions)
Phusion polymerase, by New England Biolabs (2 mentions)
Gateway cloning, by Thermo Fisher Scientific (2 mentions)
Bp recombination reaction, by Thermo Fisher Scientific (2 mentions)
Gateway system, by Thermo Fisher Scientific (2 mentions)
Gibson cloning, by New England Biolabs (2 mentions)
106 protocols using pdonr207
1
Gateway Cloning of Ern1 and Rap2.11
2
Cloning SUB ORF into Yeast Two-Hybrid Vector
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SUB-encoding open reading frame (ORF) was amplified by standard PCR (Platinum taq; Invitrogen, Carlsbad, CA, USA) using sub-specific primers flanked with the Gateway cloning sites 5′-GGGGACAACTTTGTACAAAAAAGTTGGC and 5′-GGGGACAACTTTGTACAAGAAAGTTGG. PCR products were cloned by in vitro recombination (Gateway System BP cloning reaction; Invitrogen) into pDONR207 (Invitrogen). The sub ORF was then transferred from pDONR207 into the Gal4-BD yeast two-hybrid vector pDEST32 according to the manufacturer’s recommendations (LR cloning reaction; Invitrogen).
3
Yeast-Based Complementation Assay for LHT1 Transporters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare yeast expression constructs, the previously described pENTR/D-TOPO::PtrLHT1.2 vector was used. As positive control, AtLHT1 was amplified with AtLHT1_cloning primers (Table S2 available as Supplementary data at Tree Physiology Online. The fragment was introduced into pDONR207 (Invitrogen) by recombination and the resulting vector pDONR207::AtLHT1 was sequenced. In a second recombination step, both genes were cloned into pDRf1-GW (Loque et al. 2007 (link)) using LR Clonase II (Invitrogen). As negative control pDRf1-GW without respective insert was chosen.
Saccharomyces cerevisiae strain 22574d (Jauniaux et al. 1987 (link)) was a gift from Prof. Dr B. André and was transformed with the three constructs according to the LiAc method (Ito et al. 1983 (link)). Positive clones were selected on medium, lacking uracil. Single colonies were selected for complementation studies. Complementation was performed on medium containing yeast N base without AAs and without ammonium sulfate, but which was supplemented with either 3 mM L-proline, L-citrulline or 3 mM GABA. As growth control, yeast was plated on 10 mM ammonium sulfate-containing medium. Non-supplemented medium served as negative control (Hirner et al. 2006 (link)). Pictures were taken after incubation at 30 °C for 10 days. The experiment was repeated three times and, one representative picture is shown.
Saccharomyces cerevisiae strain 22574d (Jauniaux et al. 1987 (link)) was a gift from Prof. Dr B. André and was transformed with the three constructs according to the LiAc method (Ito et al. 1983 (link)). Positive clones were selected on medium, lacking uracil. Single colonies were selected for complementation studies. Complementation was performed on medium containing yeast N base without AAs and without ammonium sulfate, but which was supplemented with either 3 mM L-proline, L-citrulline or 3 mM GABA. As growth control, yeast was plated on 10 mM ammonium sulfate-containing medium. Non-supplemented medium served as negative control (Hirner et al. 2006 (link)). Pictures were taken after incubation at 30 °C for 10 days. The experiment was repeated three times and, one representative picture is shown.
4
Gateway Cloning of TSWV NSs-GFP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TSWV NSs coding sequence (CDS) was previously described [8 (link)]. The NSs CDS was amplified using the PCR primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTCTTCAAGTGTTTATGAG-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGTTTTGATCCTGAAGCATA-3′. The amplified NSs CDS was cloned into the Gateway Donor vector pDONR 207 (Invitrogen) via a BP reaction (insertion of the att-B-sequence-containing PCR product into the att P recombination sites) and then inserted into the destination expression vector pEarleyGate 103 [44 (link)] from pDONR 207 via an LR reaction (insertion of the att-L-sequence-containing DNA into the att R recombination sites). In pEarleyGate 103, NSs was fused with an mGFP5 tag at its C-terminal (NSs-GFP). All the PCR-amplified sequences used in this research were verified by sequencing.
5
Overexpression and Binding Analysis of CaCBL1 and CaWRKY40
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze CaCBL1 transient overexpression, the ORF of CaCBL1 with or without termination codon was cloned into the entry vector pDONR207 (Invitrogen) by BP reaction and was then cloned into destination vectors such as pEarleyGate103 (Invitrogen) with a GFP protein tag for subcellular localization or pEarleyGate202 (Invitrogen) harboring a Flag protein tag for ChIP assay by LR reaction.
To investigate the possible binding of CaWRKY40 to W-box-2 within the CaCBL1 promoter, the ORF of CaWRKY40 or its mutant (CaWRKY40-m) was cloned into the vector pDONR207 by BP reaction and was then cloned into destination vector pEarleyGate202 for ChIP assay or pDEST15 (harboring a GST protein tag) for EMSA by LR reaction.
To generate constructs for a VIGS assay, a specific DNA fragment of CaCBL1 in its ORF was cloned into the entry vector pDONR207 by BP reaction and was then cloned into destination vector pTRV2 (Invitrogen) by LR reaction. The primers used for vector constructions of CaCBL1 and CaWRKY40 in this study are listed in Supplementary Table S1.
The vectors of CabZIP63 and CaWRKY40 used in this study were previously constructed (Shen et al. 2016a ). The vectors construction followed Gateway Recombination Cloning Technology (Invitrogen Corp.).
To investigate the possible binding of CaWRKY40 to W-box-2 within the CaCBL1 promoter, the ORF of CaWRKY40 or its mutant (CaWRKY40-m) was cloned into the vector pDONR207 by BP reaction and was then cloned into destination vector pEarleyGate202 for ChIP assay or pDEST15 (harboring a GST protein tag) for EMSA by LR reaction.
To generate constructs for a VIGS assay, a specific DNA fragment of CaCBL1 in its ORF was cloned into the entry vector pDONR207 by BP reaction and was then cloned into destination vector pTRV2 (Invitrogen) by LR reaction. The primers used for vector constructions of CaCBL1 and CaWRKY40 in this study are listed in Supplementary Table S1.
The vectors of CabZIP63 and CaWRKY40 used in this study were previously constructed (Shen et al. 2016a ). The vectors construction followed Gateway Recombination Cloning Technology (Invitrogen Corp.).
6
Generating Chimeric Constructs via Gibson Assembly
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Domain swap constructs were generated using Gibson assembly Master Mix (New England BioLabs).
The coding sequences of RLP42 and RLP40 were cloned into pDONR207 (Invitrogen) and/or pLOCG vector 36 (link) . Using the resulting plasmids as templates, the corresponding fragments were amplified with overlap regions and assembled with SpeI-digested pLOCG to generate the chimeric constructs with Cterminal GFP fusion. Mutation constructs were generated either using Gibson assembly Master Mix as described above or the GeneArt Site-Directed Mutagenesis System (Thermo Scientific) and AccuPrime Pfx DNA Polymerase (Thermo Scientific) using pDONR207::RLP42 as template. Mutated RLP42 sequences were recombined into pB7FWB2.0 (Plant Systems Biology, VIB, University of Ghent) for Cterminal GFP fusion. Primers are listed in Table S1.
The coding sequences of RLP42 and RLP40 were cloned into pDONR207 (Invitrogen) and/or pLOCG vector 36 (link) . Using the resulting plasmids as templates, the corresponding fragments were amplified with overlap regions and assembled with SpeI-digested pLOCG to generate the chimeric constructs with Cterminal GFP fusion. Mutation constructs were generated either using Gibson assembly Master Mix as described above or the GeneArt Site-Directed Mutagenesis System (Thermo Scientific) and AccuPrime Pfx DNA Polymerase (Thermo Scientific) using pDONR207::RLP42 as template. Mutated RLP42 sequences were recombined into pB7FWB2.0 (Plant Systems Biology, VIB, University of Ghent) for Cterminal GFP fusion. Primers are listed in Table S1.
7
Cloning and Expression of Effector and SUMO Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The coding sequence of Mp64, lacking the region encoding the N-terminal signal peptide, was amplified from M. persicae (JHI_genotype O) cDNA by PCR with gene-specific primers DONR-Mp64_F and DONR-Mp64_Rev (Supplementary Table S2.) The amplicon was cloned into entry vector pDONR207 (Invitrogen) using Gateway cloning technology. Cloning of the Phythophthora capsici effector CRN83_152 and the CRN83_152_6D10 mutant was previously described (Stam et al., 2013; Amaro et al., 2018) . For in planta expression, both effectors were cloned into destination vector pB7WGF2 (N-terminal GFP tag) (Karimi et al., 2002) .
An entry clone carrying AtSIZ1 was kindly provided by Dr H.A. van den Burg, The University of Amsterdam. NbSIZ1 was amplified from N. benthamiana cDNA with gene-specific primers NbSIZ1-attB1 and NbSIZ1-attB2 or NbSIZ1-attB2-nostop (Supplementary Table S2). Amplicons were cloned into entry vector pDONR207 (Invitrogen) using Gateway technology. For in planta expression, AtSIZ1 and NbSIZ1 were cloned into destination vectors pB7FWG2 (C-terminal GFP tag) (Karimi et al., 2002) , pK7RWG2 (C-terminal mRFP tag, Karimi et al., 2005) , and pGWB20 (Cterminal 10xMyc tag) (Nakagawa et al., 2007) .
An entry clone carrying AtSIZ1 was kindly provided by Dr H.A. van den Burg, The University of Amsterdam. NbSIZ1 was amplified from N. benthamiana cDNA with gene-specific primers NbSIZ1-attB1 and NbSIZ1-attB2 or NbSIZ1-attB2-nostop (Supplementary Table S2). Amplicons were cloned into entry vector pDONR207 (Invitrogen) using Gateway technology. For in planta expression, AtSIZ1 and NbSIZ1 were cloned into destination vectors pB7FWG2 (C-terminal GFP tag) (Karimi et al., 2002) , pK7RWG2 (C-terminal mRFP tag, Karimi et al., 2005) , and pGWB20 (Cterminal 10xMyc tag) (Nakagawa et al., 2007) .
8
JAGGER Promoter and Overexpression Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A DNA fragment encoding the promoter region of JAGGER was amplified by PCR using the primers AtP_4390 and AtP_4391 (Supplemental Table 1), including 3051 bp upstream of the 5 0 untranslated region. The amplified fragment was cloned into pDONR207 TM (Invitrogen). The promoter fragment was transferred into the binary vector pBGWFS7 (Karimi et al., 2002) to obtain the JAGGER prom :GUS construct. An overexpression vector was obtained using a 763-bp DNA fragment corresponding to the JAGGER-coding sequence. This DNA fragment was amplified by PCR using the primers AtP_4486 and AtP_4487 (Supplemental Table 1). The amplified fragment was cloned into pDONR207 TM (Invitrogen) and thereafter transferred into the overexpression vector pB2GW7 (Karimi et al., 2002) to obtain the 35s prom :JAGGER construct. To construct pSTK:JAGGER_RNAi, we amplified a specific JAGGER fragment (231 bp) using primers AtP_4339 and AtP_4340, and recombined into RNAi vector pFGC5941 (Karimi et al., 2002) through an LR reaction (Invitrogen). The CaMV 35S promoter of the pFGC5941 vector was removed and substituted by the STK promoter (amplified using primers AtP_590 and AtP_591) (Kooiker et al., 2005) . All constructs were confirmed by DNA sequencing. A. thaliana Col-0 was transformed by the floral dip method (Clough and Bent, 1998) .
9
Cloning and Expression of SPL-GFP Fusion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Initially, the SPL locus was cloned into pDONR207 (Life Technologies) and subsequently transferred to the pBGWFS7 destination vector (ThermoFisher Scientific); the expression vector was used to amplify the SPL genomic region fused to the GFP reporter gene. The fragment obtained was cloned into pDONR207 (Life Technologies). The putative promoter region of the gene plus the 5′ UTR and the 3′ UTR were cloned and subsequently transferred to pDONR201 P4-P1r and pDONR221 P2r-P3, respectively. By a multisite gateway approach, we obtained pSPL:SPL_GFP-3′UTR, combining the obtained donor vectors (described above) and transferring them into the pH7M34GW destination vector (ThermoFisher Scientific). The primers used are listed in Table S5 .
10
Evaluating Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the ability of ERA to interact with other partners, the coding sequences of ERA, INCO and SQUA were cloned into pDONR207 (Life Technologies) and subsequently by Gateway recombination transferred to the GAL4 system (pGADT7 and pGBKT7; Clontech) or pTFT1 Gateway (kindly provided by Marcos Egea Cortines; ref. 44 (link)). The two- and three-hybrid assays were performed at 28 °C in the yeast strain AH109 (Clontech) and were tested on selective yeast synthetic dropout medium lacking leucine, tryptophan, adenine and histidine and supplemented with different concentrations of 3-aminotriazole (1, 2 and 5 mM of 3-AT). For BiFC assay, the coding sequences of ERA, INCO and SQUA cloned into pDONR207 (Life Technologies) were transferred by Gateway recombination to the pYFPN43 and pYFPC43 vectors. BiFC was performed as previously described by Belda-Palazon et al.30 using in the co-infiltration procedure the p19 protein of the tomato bushy stunt virus to suppress gene silencing. The abaxial surfaces of infiltrated tobacco leaves were analyzed 5 days after inoculation. Primers used for gene cloning are reported in Supplementary Table S1 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!