C. elegans expression plasmids utilized the Pmec-7 promoter from the mec-7 gene for touch neuron expression[68 (link)]. Vector details are available upon request. Cloning was performed using the Gateway in vitro recombination system (Invitrogen, Carlsbad, CA) using Grant lab-modified versions of MiniMos enabled vectors pCFJ1662 (Hygromycin resistant; Addgene #51482) and pCFJ910 (G418 resistant; Addgene #44481) (gifts of Erik Jorgensen, University of Utah): pCFJ1662 Pmec-7 GTWY mNeonGreen let858 (34F6); pCFJ1662 Pmec-7 mNeonGreen GTWY let858 (34D4); pCFJ1662 Pmec-7 GTWY oxGFP let858 (36G3); pCFJ910 Pmec-7 mScarleti GTWY let858 (33B6); and pCFJ910 pmec-7 GTWY mScarleti (35D2). pDONR221 entry vectors containing coding regions for rab-7, lmp-1, lgg-1, clic-1, snx-1, rme-8, were recombined into neuronal destination vectors by Gateway LR clonase II reaction to generate C-/N- terminal fusions. Single-copy integrations were obtained by MiniMOS technology [69 (link)].
Pcfj910
Manufactured by Addgene
The PCFJ910 is a lab equipment product. It is a compact, benchtop instrument designed for performing polymerase chain reaction (PCR) experiments. The device features a temperature-controlled block that can accommodate multiple samples simultaneously. The PCFJ910 allows for precise control of the thermal cycling parameters required for DNA amplification during PCR protocols.
Automatically generated - may contain errors
Lab products found in correlation
Pcfj1662, by Addgene (2 mentions)
Ppd49, by Addgene (2 mentions)
Pcfj68, by Addgene (2 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Nebuilder system, by New England Biolabs (1 mentions)
Pma122, by Addgene (1 mentions)
Nebbuilder hifi dna assembly, by New England Biolabs (1 mentions)
6 protocols using pcfj910
1
Molecular Cloning of C. elegans Reporters
2
Gateway and Gibson Assembly for Single-Copy C. elegans Transgenics
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Plasmids for C. elegans coleomocyte expression were produced by standard methods including in vitro recombination via the Gateway system (ThermoFisher) and/or Gibson Assembly using the Nebuilder system (NEB). Plasmid backbones were pCFJ1662 and pCFJ910 (Addgene) and used promoters from the snx-1 or cup-4 genes. Our minimos protocol for single copy transgene integration is based on a protocol found on wormbuilder.org as described in [39 (link)]. The gonad arms of first day gravid adults were microinjected with plasmid mixtures including 10ng/ul drug resistant expression plasmid (G418 or Hygromycin) [40 (link),41 (link)] 10ng/μl pGH8, 2.5ng/μl pCFJ90, 65ng/μl pCFJ601, and 10ng/ul pMA122. 2–3 injected animals per plate were incubated at 25°C, with selection drug added between 24–72 hrs post injection. Plates were screened for single copy integrated transformants after a minimum of 10 days of growth, focused on populations that survive drug selection and lack extrachromosomal arrays visualized with the mCherry co-injection markers. Candidate single-copy integrants were passaged on drug containing plates and analyzed for expression. Lines displaying 100% transmission of the expressed transgene without drug selection were frozen and used for experiments.
3
Single-Copy Insertions of EGL-19B Fluorescent Fusion
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KP#3308 has the myo-3 promoter, PAT-4 signal sequence (from pPD122.36 Addgene), super-ecliptic pHluorin (Dittman and Kaplan, 2006 (link)), PAT-3 transmembrane domain (pPD122.36) followed by the cDNA of EGL-19B coding for residues 1374–1872, tagRFP-T (codon optimized for C. elegans), and the cDNA of EGL-19B coding for residues 1873–1877, followed by the let-858 3’ UTR (pPD122.36) inserted between the HindIII and AflII sites in the polylinker of the miniMOS vector pCFJ910 (Addgene).
Single copy insertions of Terrier were obtained using the miniMOS technique as described (Frøkjær-Jensen et al., 2014 (link)).
Single copy insertions of Terrier were obtained using the miniMOS technique as described (Frøkjær-Jensen et al., 2014 (link)).
4
Standard Molecular Cloning Protocols
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Standard restriction digestion or PCR based cloning was performed for all the experiments in this study (Russell, 2001 ). The vectors used in this study are pPD49.26, PCFJ910 and PCFJ68 all obtained from Addgene. See also KRT table . A detailed description of all the constructs generated for the study and their respective cloning primers are indicated in the supplemental section Table S1 .
5
Construction of Inducible Cas9 System
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Strains generated for this publication along with key plasmids and reagents are listed in the Key Resources Table. A full list of all plasmids is given in Supplementary file 1 . All plasmids were cloned by Gibson Assembly following the standard NEB Builder HiFi DNA Assembly master mix protocol (New England Bio Labs [NEB], MA), unless otherwise indicated. All plasmids have been confirmed by restriction digest, Sanger sequencing, and/or full plasmid sequencing. All primers used in the construction and validation of plasmids are listed in Supplementary file 2 .
To generate our heatshock-inducible Cas9, hsp16.41p::Cas9dpiRNA::tbb-2 ′3UTR, the hsp16.41 promoter was amplified from pMA122 (Addgene ID34873) (Frøkjær-Jensen et al., 2012 (link)). The germline-licensed Cas9 and tbb-2 3′ UTR were amplified from pCFJ150-Cas9 (dpiRNA) (Addgene ID107940) (Zhang et al., 2018 (link)). All fragments were assembled into PCR-linearized pUC19 vector (NEB) to give the final plasmid pZCS36.
To generate a standard empty guide vector, U6p::(empty)gRNA, the U6p and gRNA scaffold from pDD162 (Addgene ID47549) (Dickinson et al., 2015 (link)) was amplified and assembled into PCR-linearized pUC19 to generate pZCS11.
To generate rsp-27p::NeoR::unc-54 3′ UTR, the full resistance cassette was amplified from pCFJ910 (Addgene ID44481) and assembled into PCR-linearized pUC19 vector to give pZCS38.
To generate our heatshock-inducible Cas9, hsp16.41p::Cas9dpiRNA::tbb-2 ′3UTR, the hsp16.41 promoter was amplified from pMA122 (Addgene ID34873) (Frøkjær-Jensen et al., 2012 (link)). The germline-licensed Cas9 and tbb-2 3′ UTR were amplified from pCFJ150-Cas9 (dpiRNA) (Addgene ID107940) (Zhang et al., 2018 (link)). All fragments were assembled into PCR-linearized pUC19 vector (NEB) to give the final plasmid pZCS36.
To generate a standard empty guide vector, U6p::(empty)gRNA, the U6p and gRNA scaffold from pDD162 (Addgene ID47549) (Dickinson et al., 2015 (link)) was amplified and assembled into PCR-linearized pUC19 to generate pZCS11.
To generate rsp-27p::NeoR::unc-54 3′ UTR, the full resistance cassette was amplified from pCFJ910 (Addgene ID44481) and assembled into PCR-linearized pUC19 vector to give pZCS38.
6
Standard Molecular Cloning Protocols
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