The largest database of trusted experimental protocols

Gateway lr recombination reaction

Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom

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.

Automatically generated - may contain errors

25 protocols using gateway lr recombination reaction

1

Yeast Two-Hybrid Analysis of GI Interactions

Check if the same lab product or an alternative is used in the 5 most similar protocols
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)).
+ Open protocol
+ Expand
2

Arabidopsis Circadian Rhythm Reporter Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
3

HvCCA1 Overexpression in Arabidopsis

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
4

Yeast Two-Hybrid Assay for GI Interactions

Check if the same lab product or an alternative is used in the 5 most similar protocols
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)).
+ Open protocol
+ Expand
5

Gateway Cloning of LEA5-YFP

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
6

Comparative analysis of ZRS enhancer

Check if the same lab product or an alternative is used in the 5 most similar protocols
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).
+ Open protocol
+ Expand
7

Cloning and Transformation of AaMYB17 in A. annua

Check if the same lab product or an alternative is used in the 5 most similar protocols
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)).
+ Open protocol
+ Expand
8

Gateway Cloning of CaMKII Isoforms

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
9

In vitro protein interaction assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
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).
+ Open protocol
+ Expand
10

Comparative analysis of ZRS enhancer

Check if the same lab product or an alternative is used in the 5 most similar protocols
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).
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!