Multisite gateway

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Multisite Gateway is a laboratory equipment product that serves as a central communication hub for connecting multiple analytical instruments and devices. It facilitates the exchange of data and control signals between these connected systems, enabling seamless integration and coordination within the laboratory environment.

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Lab products found in correlation

Phusion polymerase, by Thermo Fisher Scientific (1 mentions) Pgem t easy, by Promega (1 mentions) Bp clonase, by Thermo Fisher Scientific (1 mentions) Primestar polymerase, by Takara Bio (1 mentions) Pentr d topo, by Thermo Fisher Scientific (1 mentions) Gateway lr clonase mix, by Thermo Fisher Scientific (1 mentions) Platinum pfx, by Thermo Fisher Scientific (1 mentions) Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions) Sanger sequencing, by Genewiz (1 mentions) Epifluorescence microscope, by Leica (1 mentions) Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions) Gibson assembly cloning kit, by New England Biolabs (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Lr clonase 2 plus enzyme, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Multisite Gateway technology (39 citations) Multisite Gateway system (35 citations) MultiSite Gateway Three-Fragment Vector Construction Kit (25 citations) Multisite Gateway Pro (5 citations) Multisite Gateway Manual (5 citations) Multisite Gateway recombination (4 citations) MultiSite Gateway Kit (4 citations) Tol2 MultiSite Gateway® kit (2 citations) Gateway Multisite Cloning kit (2 citations) Multisite Gateway manual Version D, 2007 (2 citations)

29 protocols using multisite gateway

Creating pENTR-PpU6-sgRNA Vectors

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To generate pENTR‐PpU6‐sgRNA‐L1L2, the first Gateway entry vector for the P. patens vector system, we amplified the PpU6 and sgRNA fragments with two separate PCRs. For the PpU6 fragment, we used a forward primer containing an AscI site and a reverse primer containing two inverted BsaI sites at the 5′ ends (Table S1). Similarly, for the sgRNA fragment we used a forward primer containing two inverted BsaI sites and a reverse primer containing a SalI site (Table S1). The two fragments were then ligated using an overlap extension PCR and ligated into pGEM/T‐Easy (Promega). Positive clones were digested with AscI and SalI, and the dropout was subsequently ligated into an AscI‐ and SalI‐linearized pENTR‐PpU6‐sgRNA‐NGG plasmid.
To generate the six entry vectors compatible with Multisite Gateway (Invitrogen) for multiplexing experiments, we amplified the sgRNA expression cassette from pENTR‐PpU6‐sgRNA‐L1L2 using primers (Table S1) containing different Multisite Gateway attachment sites (attB) and subsequently recombined with the Multisite Gateway pDONR221 plasmid set (Invitrogen) using a BP clonase reaction following the manufacturer's recommendations.

Mallett D.R., Chang M., Cheng X, & Bezanilla M. (2019). Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens. Plant Direct, 3(9), e00168.

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Lentiviral Vector Construction and Applications

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Lentiviruses used in this study (pLenti6, Invitrogen) are derived from a third-generation human immunodeficiency virus -1-based self-inactivating lentiviral vector35 (link). Lentiviral transfer vectors were constructed using the modular MultiSite Gateway cloning technology (Invitrogen) to generate pLenti6/UbC-EGFP, pLenti6/CMV-ZsGreen, pLenti6/CMV-H2B-GFP, pLenti6/UbC-mCherry and pLenti/CMV-SV40er. Detailed cloning information is provided in the Supplementary Materials.

Hines W.C., Yaswen P, & Bissell M.J. (2015). Modelling breast cancer requires identification and correction of a critical cell lineage-dependent transduction bias. Nature Communications, 6, 6927.

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Multisite Gateway Assembly of Fluorescent Reporters

Cited 2 times

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Pmex-5::nmy-2-yfp::nos-2^3′UTR was assembled from plasmid Pmex-5::hmr-1-gfp::nos-2^3′UTR, which was constructed using Multisite Gateway (Invitrogen) from vector pCFJ150 (Addgene #19329) (Frøkjær-Jensen et al., 2008 (link)), 5′ entry clone pJA252 (Pmex-5; Addgene #21512; Zeiser et al., 2011 (link)), middle entry clone pJN527 (hmr-1-gfp; Chihara and Nance, 2012 (link)), and 3′ entry clone pDC10 (nos-2 3′ UTR; Chihara and Nance, 2012 (link)). hmr-1-gfp was replaced with nmy-2 genomic sequence and yfp in a three-fragment Gibson Assembly reaction (Gibson et al., 2009 (link)).
Pmex-5::zen-4-yfp::nos-2^3′UTR was constructed from Pmex-5::nmy-2-yfp::nos-2^3′UTR, replacing nmy-2 with zen-4 genomic sequence via Gibson Assembly.
Ppie-1::gfp-Moesin::nos-2^3′UTR was constructed from vector pKS111-His (Ppie-1::gfp-Histone::nos-2^3′UTR; a gift from Kuppuswamy Subramaniam; D’Agostino et al., 2006 (link)). pKS111-His was digested with SpeI to remove the Histone H2B coding region. cDNA encoding amino acids 438–575 of Drosophila melanogaster Moesin (isoform D) was amplified by PCR using primers containing SpeI restrictions sites and ligated with the pKS111-His vector fragment.

Maniscalco C., Hall A.E, & Nance J. (2019). An interphase contractile ring reshapes primordial germ cells to allow bulk cytoplasmic remodeling. The Journal of Cell Biology, 219(2), e201906185.

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Generating C. elegans Transgenic Strains

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Ten HOT core sites were PCR amplified from N2 genomic DNA using Phusion polymerase (Finnzymes) and Gateway cloned (Invitrogen) into pDONR221 (regions given in Supplemental Table S1). To create transgenes to test whether HOT regions could function as promoters, MultiSite Gateway cloning (Invitrogen) was used to recombine the HOT regions upstream of his-58 (pJA272) and gfp∷tbb-2 3′UTR (pJA256) sequences on the MosSCI compatible vector pCFJ150, which targets Mos site Mos1(ttTi5605) chrII (Zeiser et al. 2011 (link)). C. elegans MosSCI lines were generated as described (Frøkjær-Jensen et al. 2008 (link)), injecting strain EG6699 with injection mixes that contained pCFJ103(40 ng/µL), pCFJ90(5 ng/µL), pCFJ104(2.5 ng/µL), and expression clones at 40 ng/µL. Supplemental Table S2 lists all strains generated in this study. All strains were used and cultured using standard methods (Brenner 1974 (link)). Transgenic strains were grown at 25°C prior to microscopic examinations. Young adult or L4 stage worms were sedated in 5 mM Tetramisole, aligned, and scanned in groups at controlled laser and scanning settings. A full list of primers used for generation of pDONR221 promoter constructs can be found in Supplemental Table S3.

Chen R.A., Stempor P., Down T.A., Zeiser E., Feuer S.K, & Ahringer J. (2014). Extreme HOT regions are CpG-dense promoters in C. elegans and humans. Genome Research, 24(7), 1138-1146.

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Zebrafish Melanoma Model for Oncogene Testing

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Zebrafish develop melanoma when melanocytes express human oncogenic Braf-V600E in a tp53-deficient
background (Patton et al., 2005 (link)). When crossed to a mitfaloss-of-function mutant, melanocyte development is impaired and melanoma no longer form. Injection of the transposon-based
expression vector MiniCoopR (MCR) into
Tg(mitfa:BRAF^V600E);tp53^−/−;mitfa^−/−one-cell stage zebrafish embryos rescues melanocytes by restoring mitfa, and simultaneously allows the
expression of a candidate gene in the rescued pigment lineages (Ceol et al., 2011 (link)). MCR
expression constructs were created by MultiSite Gateway recombination (Invitrogen) using full-length human open reading
frames. Briefly, 25 pg of MCR:LSD1 or MCR:EGFP were microinjected together with 25 pg of Tol2 transposase mRNA into one-cell
Tg(mitfa:BRAF^V600E);tp53^−/−;mitfa^−/−zebrafish embryos. Embryos were scored for melanocyte rescue at 48–72 hours post-fertilization, and equal numbers were
raised to adulthood, and scored weekly (8–12 weeks) or biweekly (> 12 weeks) for the emergence of melanoma lesions.
Zebrafish were maintained under Institutional Animal Care and Use Committee-(IACUC)-approved conditions.

Yu Y., Schleich K., Yue B., Ji S., Lohneis P., Kemper K., Silvis M.R., Qutob N., van Rooijen E., Werner-Klein M., Li L., Dhawan D., Meierjohann S., Reimann M., Elkahloun A., Treitschke S., Dörken B., Speck C., Mallette F.A., Zon L.I., Holmen S.L., Peeper D.S., Samuels Y., Schmitt C.A, & Lee S. (2018). Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma. Cancer cell, 33(2), 322-336.e8.

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Genetic Constructs for Auxin Signaling Visualization in Brachypodium

Cited 1 time

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All constructs were cloned using Multi-site Gateway (Invitrogen Grand Island, NY) and were transformed into Brachypodium Bd21-3 using previously published methods (Bragg et al., 2015 (link)). For pZmUbi::DII-Venus, we first cloned the maize ubiquitin promoter into pDONR P4-P1R (Primer IDs 9–10 Table 1) and this was subsequently recombined with pDONR 221 containing Arabidopsis DII and pDONR P2R-P3 containing Venus-N7 (Brunoud et al., 2012 (link)) into the Multi-site Gateway binary vector pH7m34GW (http://gateway.psb.ugent.be/). In the T3 generation degradation of DII-Venus in the presence of auxin was validated by treating excised Brachypodium spikelet meristems with 1 µM 1-naphthaleneacetic acid (NAA) or mock treatment in 70% ethanol and imaging every 30 min (Figure 1—figure supplement 2).
For SoPIN1-Cerulean, the promoter plus 5’ coding pDONR-P4-P1R and 3’ coding plus downstream pDONR-P2R-P3 fragments from (O'Connor et al., 2014 (link)) were used. Maize codon-optimized Cerulean fluorescent protein, courtesy of David Jackson, was amplified with 5x Ala linkers and cloned into pENTR/D-TOPO. These three fragments were then recombined into pH7m34GW.

O'Connor D.L., Elton S., Ticchiarelli F., Hsia M.M., Vogel J.P, & Leyser O. (2017). Cross-species functional diversity within the PIN auxin efflux protein family. eLife, 6, e31804.

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Generating Transgenic Constructs and Plants

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Transgenic constructs were generated using the Gateway or MultiSite Gateway system (Invitrogen) and were introduced into plants using the floral dip method51 (link). For pPLM::PLM-YFP, a 2.2-kb genomic DNA fragment with a 1.5-kb upstream region was amplified and combined with YFP into the MultiSite destination vector pBm43GW. Analysis was carried out in the F2 or F3 generations of crosses with wild type or mutant plants. Primer sequences are listed in Supplemental Table 1.

Yan D., Yadav S.R., Paterlini A., Nicolas W.J., Petit J.D., Brocard L., Belevich I., Grison M.S., Vaten A., Karami L., el-Showk S., Lee J.Y., Murawska G.M., Mortimer J., Knoblauch M., Jokitalo E., Markham J.E., Bayer E.M, & Helariutta Y. (2019). Sphingolipid biosynthesis modulates plasmodesmal ultrastructure and phloem unloading. Nature plants, 5(6), 604-615.

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Generating Transgenic Zebrafish Lines

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Cloning of the zebrafish dβh promoter (5.2 kb) and creation of dβh:EGFP and dβh:MYCN lines have been previously described (13 (link)). Similarly, mCherry and human c-MYC cDNAs were PCR cloned into the pENTR223 vector, and dβh:mCherry and dβh:c-MYC constructs were individually assembled by Multisite Gateway cloning (Invitrogen). Using a co-injection strategy with I-SceI meganuclease, one-cell embryos were injected with DNA constructs and grown to adulthood. Primary injectants were screened for germline transmission, and stable lines were generated by outcrossing to the wild-type AB strain.

Zimmerman M.W., Liu Y., He S., Durbin A.D., Abraham B.J., Easton J., Shao Y., Xu B., Zhu S., Zhang X., Li Z., Weichert-Leahey N., Young R.A., Zhang J, & Look A.T. (2017). c-MYC drives a subset of high-risk pediatric neuroblastomas and is activated through mechanisms including enhancer hijacking and focal enhancer amplification. Cancer discovery, 8(3), 320-335.

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Genetic Manipulation Constructs for Epichloë Fungi

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Gene deletion constructs for dmaW, easG, cloA, and lpsB were created by means of MultiSite Gateway cloning (Invitrogen) and utilized a split marker system [39 (link)] for improved homologous recombination frequencies. Flanking sequences for each gene were amplified by PCR with attB1- and attB2-tailed primers (Supplementary Figure S1 and Supplementary Table S1 from E. Festucae var. lolii AR5 genomic DNA. These fragments were recombined into pDONR SML and pDONR SMR vectors using BP clonase (invitrogen). Linear PCR products used for transformation were amplified using PrimeSTAR polymerase (Takara, Shiga, Japan) and primers listed in Supplementary Table S1.

Hudson D., Mace W., Popay A., Jensen J., McKenzie C., Cameron C, & Johnson R. (2021). Genetic Manipulation of the Ergot Alkaloid Pathway in Epichloë festucae var. lolii and Its Effect on Black Beetle Feeding Deterrence. Toxins, 13(2), 76.

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Generating Transgenic Constructs and Plants

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