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Multisite gateway pro

Manufactured by Thermo Fisher Scientific
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The Multisite Gateway Pro is a versatile laboratory instrument designed to facilitate automated sample processing. It enables the simultaneous handling of multiple samples across different workflows, increasing efficiency and throughput in the laboratory environment.

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Lab products found in correlation

Pentr d topo vector, by Thermo Fisher Scientific (1 mentions) Phic31 integration, by BestGene (1 mentions) Quick ligation kit, by New England Biolabs (1 mentions) Nebuilder hifi dna assembly, by New England Biolabs (1 mentions) Primestar max, by Takara Bio (1 mentions)

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Multisite Gateway (29 citations) MultiSite Gateway Pro Kit (2 citations)

5 protocols using multisite gateway pro

1

Synthetic 10-plex Multicopy Array Construction

Cited 1 time
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Synthetic 10-plex MC array constructs were synthesized by GenScript and moved into pMME977 (thyA+) by Multisite Gateway Pro cloning (Invitrogen 12537–100) as previously described17 (link). The MC arrays tested here bear a boxA sequence −58 bp upstream of the first repeat which was added after vector completion by quick-change PCR as described previously17 (link). Final plasmids were introduced to Lp02(dcas9) by electroporation and the strains containing the plasmids were selected for on CYE plates43 (link) without thymidine. Spacer composition of each array and their corresponding nucleotide sequences are listed in Table S1.
Ellis N.A., Myers K.S., Tung J., Ward A.D., Johnston K., Bonnington K.E., Donohue T.J, & Machner M.P. (2023). A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes. bioRxiv.
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2

Overexpression of EIN3 and ORA59

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Overexpression constructs were prepared by amplifying EIN3 (AT3G20770) and ORA59 (AT1G06160) from the cDNA of Wild type using listed primers (Supplementary Table 1), followed by cloning the genes into pENTR™-D-TOPO vector (Invitrogen), and subsequent recombination with pEN-L4-p35S-R1 and pEN-R2-Luc-L3 using multiple LR reactions together with pB7m34GW as a destination vector according to MultiSite Gateway® Pro (Invitrogen). The resulting binary vectors contained 35S:ORA59:LUC and 35S:EIN3:LUC sequences were used to transform Wild type and ein3 eil1 by the floral dip method (Clough and Bent 1998 (link)).
He X., Jiang J., Wang C, & Dehesh K. (2017). ORA59 and EIN3 interaction couples jasmonate-ethylene synergistic action to antagonistic salicylic acid regulation of PDF expression. Journal of integrative plant biology, 59(4), 275-287.
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3

Constructing mEGFP-PpCESA Fusion Vectors

Cited 2 times
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Vectors for expressing mEGFP-PpCESA fusion proteins were constructed using Multisite Gateway Pro according to the manufacturer’s instruction (Invitrogen, Grand Island, NY, USA). A pDONR P1-P5r entry clone containing the coding sequence for mEGFP47 (link) was linked to a pDONR P5-P2 entry clone containing the coding sequence of PpCESA548 (link) and inserted into the pTHUbiGate destination vector, which drives expression with the maize Ubiquitin promoter49 (link). The P1-P5r mEGFP entry clone was linked to a pDONR P5-P2 entry clone containing the coding sequence of PpCESA833 (link) and inserted into the pTHAct1Gate destination vector, which drives expression with the rice Actin1 promoter50 (link). By using different destination vectors, we were able to test two constitutive promoters for use in this application. Both vectors confer hygromycin resistance and target the expression cassette to the intergenic 108 locus22 (link). Vectors were cut with SwaI for transformation into their respective knockout lines.
Tran M.L., McCarthy T.W., Sun H., Wu S.Z., Norris J.H., Bezanilla M., Vidali L., Anderson C.T, & Roberts A.W. (2018). Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens. Scientific Reports, 8, 735.
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4

Transgenic GAL4 Reporter Fly Generation

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Transgenic GAL4 reporter flies were generated using standard methods as described [14 (link)]. The 5’ and 3’ regions flanking the genes were amplified from bacterial artificial chromosomes (BACs) corresponding to the reference genome of Drosophila melanogaster [42 (link)]. MultiSite Gateway Pro recombination (Thermo Fisher) was used to assemble the 5’ and 3’ genomic regions with GAL4 in the pBGRY destination vector [43 (link)]. For Rh50-GAL4, the 5’ region included chromosome 3L: 4,907,401 to 4,910,693 and the 3’ region 3L: 4,918,898 to 4,924,642. For Ir25a-GAL4, the 5’ region included chromosome 2L: 4,835,726 to 4,834,655 and the 3’ region 3L: 4,830,990 to 4,827,634. For Ir75a-GAL4, the 5’ region included chromosome 3L: 17,829,014 to 17,817,922 and the 3’ region 3L: 17,815,667 to 17,811,236. PhiC31 integration (Best Gene, Inc.) was used to integrate the assembled GAL4 vectors into the Drosophila melanogaster genome. Two transgenic strains were made for each GAL4 in which the construct was incorporated into the second (attP40 landing site [44 (link)]) and third (attP2 landing site [45 (link)]) chromosomes.
Vulpe A., Kim H.S., Ballou S., Wu S.T., Grabe V., Gonzales C.N., Liang T., Sachse S., Jeanne J.M., Su C.Y, & Menuz K. (2021). An ammonium transporter is a non-canonical olfactory receptor for ammonia. Current biology : CB, 31(15), 3382-3390.e7.
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5

Plasmid DNA Construction Protocols

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All plasmid DNAs used in this study are shown in Supplementary Figs. 7 to 22. In the construction of plasmid DNAs, PCR amplification for cloning was carried out using PrimeSTAR Max (TaKaRa), cloning for assembling was performed using Quick ligation kit (NEB), NEBuilder HiFi DNA Assembly (NEB), and Multisite gateway Pro (Thermo Fisher Scientific).
Osakabe K., Wada N., Miyaji T., Murakami E., Marui K., Ueta R., Hashimoto R., Abe-Hara C., Kong B., Yano K, & Osakabe Y. (2020). Genome editing in plants using CRISPR type I-D nuclease. Communications Biology, 3, 648.
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