Accessions that were used to extract DNA for sequencing and cloning of specific SuSy genes were: S. lycopersicum cv Moneymaker (accession number LA2706), cv Large Red Cherry (accession number TR00018), S. neorickii G1.1601 (accession number CGN24193) and S. arcanum LA385 obtained from Tomato Genetics Resource Center in Davis, California (TGRC, http://tgrc.ucdavis.edu/ ). The cv Moneymaker was used in this study as the reference in place of cv Heinz because they both have the same SuSy1/3/4 protein sequences. Plasmid pGEM-T Easy (Promega, USA), PCR II-Blunt-TOPO (ThermoFisher Scientific, USA) were used to construct intermediate clones, while plasmid pDR195 (a gift from Dr. Wolf Frommer—Carnegie Institution for Science, USA) was used to make the expression clones. Competent cells of Escherichia coli strains DH5a and XL10-Gold were purchased from Invitrogen (USA) and Agilent (USA) respectively, whereas yeast Saccharomyces cerevisiae strain 22574d was provided by courtesy of Dr. Bruno André—Université Libre de Bruxelles, Belgium.
Xl10 gold
Manufactured by Thermo Fisher Scientific
Sourced in United States
The XL10-Gold is a high-efficiency chemically competent bacterial cell line designed for the transformation of plasmid DNA. It is suitable for various molecular biology applications, including cloning, site-directed mutagenesis, and protein expression. The XL10-Gold cells exhibit increased transformation efficiency compared to standard competent cells, enabling researchers to efficiently introduce DNA into the cells.
Automatically generated - may contain errors
Lab products found in correlation
Bl21 gold de3, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
Safire2 plate reader, by Tecan (2 mentions)
Kl2500 fiber optic light source, by Leica (2 mentions)
Pgem t easy, by Promega (1 mentions)
Fluo 3, by Thermo Fisher Scientific (1 mentions)
Gcamp6f, by Addgene (1 mentions)
Ez run protein ladder, by Thermo Fisher Scientific (1 mentions)
6 protocols using xl10 gold
1
Cloning and Sequencing of Tomato SuSy Genes
2
Cloning and Extraction of H. pylori Genome
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E. Coli strain XL10-gold (Invitrogen) was grown on Luria-Bertani (LB) agar plate or in LB liquid medium supplemented with ampicillin (100 μg/mL) and/or Kanamycin (50 μg/mL). The H. pylori 26695 genome was extracted by following the protocol of Bacteria Genomic DNA Kit (Zoman ZP301). The genome DNA was used as template for cloning CagZ (residues 2–199) and all constructs of Cagβ (Supplementary Table 3 ).
3
Plasmid-based Fluorescent Biosensors
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GCaMP6f, G-GECO 1.0 and GEM-GECO plasmids were purchased from Addgene (plasmid #40755, #32466 and #32463, respectively). pET41a and pEGFP-N1 vectors were obtained from Novagen and Clontech, respectively. XL10-Gold and BL21 (DE3) Gold cells were purchased from Invitrogen. Fluo-3 was purchased from Molecular Probes and EZ-Run protein ladder from Fisher Bioreagents. Restriction enzymes were obtained from New England Biolabs and T4 DNA ligase from Fermentas. Br2-BAPTA was purchased from Life Technologies.
4
Bacterial Expression Library Screening
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Mutations at specific residues were introduced by overlap-extension PCR. All PCR products were cut and ligated into a constitutive bacterial expression vector pNCS (Allele Biotech) or arabinose-inducible pBAD (Invitrogen). For all library construction methods, chemically competent Escherichia coli strain DH5α (BioExpress) or XL-10 Gold (Invitrogen) were transformed and grown overnight on LB/agar at 37°C and maintained thereafter at room temperature. For each round of mutagenesis, a number of colonies approximately 10-fold higher than the expected library diversity were screened to ensure full coverage. Agar plates were screened for transmitted color by eye and for fluorescence in a light-tight enclosure with a KL2500 fiberoptic light source (Leica) and a ST-8300M cooled CCD camera controlled with CCDOps software (Santa Barbara Instrument Group) on a MacBook Pro running OS 10.6.8 (Apple). 610/20 nm excitation and 645/30 nm emission filters (Chroma) were placed in the light path before the fiberoptic light guide and before the camera, respectively. Bacterial colonies of interest were patched on LB/agar plates and incubated overnight at 37°C. Lysates were extracted with B-PER II (Pierce), and spectra were obtained on a Safire II plate reader (TECAN). DNA sequences of all constructs are available upon request.
5
Plasmid Cloning and Cell Lines
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GCaMP6s and GCaMP6f plasmids were a gift from Douglas Kim (Addgene plasmid #40753 and #40755, respectively)19 (link). pET41a and pEGFP-N1 vectors were obtained from Novagen and Clontech, respectively. XL10-Gold and BL21 (DE3) Gold cells were purchased from Invitrogen. Restriction enzymes were obtained from New England Biolabs and T4 DNA ligase from Fermentas.
6
Bacterial Expression Library Screening
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