The long-arm HPRT targeting vectors pHPRT8.9-Puro(+) and pHPRT8.9-Puro(−) were constructed as previously described [7] (link). Other gene-targeting vectors, including the short-arm HPRT targeting vectors pHPRT2.2-Puro(+) and pHPRT2.2-Puro(−), were constructed using the MultiSite Gateway system (Life Technologies, Carlsbad, CA) to assemble two homology arms and a drug-resistance gene cassette, as previously described [1] (link), [27] (link). Genomic fragments for homology arms were PCR amplified using Nalm-6 genomic DNA as template with primers listed in Figures S7 and S8 in File S1 . Imperfect vectors lacking either arm of pHPRT8.9-Puro(+) and pHPRT8.9-Puro(−) were constructed as shown in Figure S9 in File S1 . Imperfect vectors lacking either arm of pHPRT2.2-Puro(+) and pHPRT2.2-Puro(−) were constructed by digestion with SacI (for 5'-arm deletion) or SalI (for 3'-arm deletion), followed by self-ligation of the linearized plasmid DNA. All the plasmid vectors were purified with Qiagen Plasmid Maxi Kits (Qiagen K.K., Tokyo) and linearized with an appropriate restriction enzyme prior to transfection [1] (link).
Multisite gateway system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Multisite Gateway system is a laboratory equipment product designed to facilitate the transfer of data and information between multiple sites or locations. Its core function is to enable the secure and efficient exchange of data across different systems and platforms, allowing for better coordination and collaboration between various laboratory operations.
Automatically generated - may contain errors
Lab products found in correlation
Plasmid maxi kit, by Qiagen (1 mentions)
Tol2 kit, by Thermo Fisher Scientific (1 mentions)
Lr clonase 2 plus, by Thermo Fisher Scientific (1 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Pcdna dest40, by Thermo Fisher Scientific (1 mentions)
Pdonr221, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (1 mentions)
Pdonr p4 p1r vector, by Thermo Fisher Scientific (1 mentions)
Bp clonase 2 enzyme mix, by Thermo Fisher Scientific (1 mentions)
Easy a high fidelity polymerase, by Agilent Technologies (1 mentions)
Lsm 880 confocal module, by Zeiss (1 mentions)
Axio observer z1 inverted microscope, by Zeiss (1 mentions)
Minibest dna fragment purification kit ver 4, by Takara Bio (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
35 protocols using multisite gateway system
1
Constructing HPRT Targeting Vectors
2
Recombinant Plasmid Construction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids used were constructed using standard molecular biology techniques employing recombinant PCR and the multisite gateway system (Life Technologies). The integrity of all cloned constructs was confirmed by DNA sequencing.
3
Monitoring Protein Degradation in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A cell line stably expressing Ub(G76V)-EGFP in HEK293T cells was used for the experiments and was kindly provided by F.-Ulrich Hartl. pEGFP-N1 was used as a control plasmid for EGFP expression only. HA-tagged Htt constructs with 20Q and 96Q stretches were kindly provided by F.-Ulrich Hartl. GFP-tagged CBP constructs or GFP-tagged bromodomain constructs were created via the MultiSite Gateway System (Life Technologies, 12537100). The construct containing the 3× bromodomain from CBP contains an NLS sequence at the N terminus (Philpott et al., 2014 (link)).
4
Transgenic Zebrafish Fluorescent Reporters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lines used in this study included flk:eGFP (zfin: s843Tg), HuC:mCherry (zfin: nia02Tg). GFAP:mCherry and fli1a:mCherry-GFP-LC3 were established using multisite gateway system (Life Technologies) and Tol2 kit21 (link). Zebrafish were maintained at 28.5 °C with a 14-hour light/10- hour dark cycle as previously described22 . Embryos were kept in incubator at 28.5 °C, and treated with 0.1 Mm 1-phenyl-2-thiourea (PTU, Sigma P5272) to inhibit pigment formation beyond 24 hours post-fertilization. All zebrafish experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China) and approved by the institutional review board of the Medical Faculty at the West China Hospital, Sichuan University.
5
Cloning and Transgenesis of Zebrafish drl Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The upstream cis-regulatory elements of the zebrafish drl gene (ENSDARG00000078004; ZDB-GENE-991213-3) were amplified from zebrafish wildtype genomic DNA and TOPO-cloned into the pENTR™ 5’-TOPO® TA Cloning® plasmid (Invitrogen) according to the manufacturer’s instructions. Subsequent cloning reactions for all used transgenesis constructs were performed with the Multisite Gateway system with LR Clonase II Plus (Life Technologies) according to the manufacturer’s instructions. Cloning details, transgenesis, transgenic zebrafish strains, and applied zebrafish techniques used in this study are outlined in Supplementary Methods . Primer sequences used for cloning and sgRNAs are outlined in Supplementary Table 1 .
6
Agrobacterium-mediated Plant Transformation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The multisite Gateway system (Invitrogen) was used for all constructs except the BZR1::2xYPET::3xHA recombineered BAC clone. See the Supplemental Material for details. Col-0 plants were transformed with Agrobacterium GV3101 carrying the various constructs using standard techniques. Segregation analyses at T2 and T3 generations were used to isolate single-insertion homozygous lines.
7
Generating tasiR-ARF insensitive ARF3a
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The MultiSite Gateway System (Invitrogen) was used to create the arf3a:arf3a and the arf3a:m-arf3a constructs. The coding sequences of arf3a (GRMZM2G030710_T01), and the arf3a regulatory regions (2.838 kb promoter and 1.059 kb 3′UTR) were amplified and cloned into pDONR entry vectors. To generate the tasiR-ARF insensitive arf3a version, mutations were introduced at the two tasiR-ARF target sites using megaprimer PCR mutagenesis. Entry clones for each of the two constructs were combined in a single MultiSite Gateway LR recombination reaction with the pTF101 Gateway-compatible maize transformation vector. Positive clones were transferred into Agrobacterium tumefaciens strain EHA101 and transformed into maize by the Iowa State University Plant Transformation Facility.
8
Constructing Engineered Endophilin Variants
9
Constructing Promoter-Gene Fusion Vectors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate the EN7::ΔARF5 and SCR::ΔARF5 constructs, DNA fragments containing the SCR (2,164 bp) or EN7 (1,226 bp) promoters (Malamy and Benfey, 1997 (link); Heidstra et al., 2004 (link); Koizumi and Gallagher, 2013 (link)) were inserted into a HindIII/KpnI-digested pMDC plant binary vector using the Gibson Assembly Cloning system (New England BioLabs). The cDNA fragment of ΔARF5 was then introduced into the vector using the GATEWAY system (Invitrogen). To generate the SCR::ΔARF5-GFP construct, DNA fragments containing the SCR promoter, ΔARF5, and GFP sequences were amplified by PCR, and then introduced into P4-P1, P1-P2, and P2-P3 entry vectors by BP reaction, respectively. These DNA fragments were inserted into the dpGreen plant binary vector by MULTISITE GATEWAY system (Invitrogen).
10
Rescuing YAP-1 Activity in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To rescue YAP-1 activity, a full genomic coding region of YAP-1 with its own 4 kb promoter was cloned into the GFP-containing pPD114.108 using a standard subcloning method. To express YAP-1 TRNs specifically, GFP fused YAP-1 was cloned into pCFJ150 under mec-4 promoter using the Multisite Gateway system (Invitrogen, Inc.). To obtain transgenic lines, each plasmid was injected into worms at 50 ng/μl with 50 ng/μl of pRF4 as a transgenic marker. To monitor the subcellular localization of YAP-1 in TRNs of wild type and worms lacking Wnt activities, mec-4p::GFP::YAP-1 was injected into the wild type and transgenes were transferred to each mutant background by mating.
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!