Plasmid Cloning and Sequence Analysis

DNA was amplified using a high fidelity enzyme (PrimeStar HS, Takara Bio Inc., Japan), or a standard enzyme (BIOTaq, Bioline, UK), and primers detailed in Additional file 1 Table S1. Amplicons were cloned using the CloneJET PCR Cloning Kit (Thermo Scientific) or the pGEM-T Easy Vector System (Promega). Purification of plasmids from E. coli was carried out following a boiling method [64 (link)] or using a commercial kit (Illustra plasmidPrep Mini Spin Kit, GE Healthcare). For plasmid profile gels, DNA was purified by alkaline lysis and separated by electrophoresis in 0.8% agarose gels with 1xTAE as described [25 (link)]. Plasmids were transferred to P. syringae by electroporation [65 ].
DNA sequences were compared and aligned using the BLAST algorithms [66 (link)], as well as the on-line MULTALIN [67 (link)] and EMBL-EBI server tools (http://www.ebi.ac.uk/Tools/msa/). The InterPro interface [68 (link)] (http://www.ebi.ac.uk/interpro/) was used to search for protein motifs. Nucleotide sequence visualization and manipulation was performed using the Artemis genome browser and ACT [69 (link)]. Primers were designed using the Primer3plus software [70 (link)].

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Bardaji L., Añorga M., Echeverría M., Ramos C, & Murillo J. (2019). The toxic guardians — multiple toxin-antitoxin systems provide stability, avoid deletions and maintain virulence genes of Pseudomonas syringae virulence plasmids. Mobile DNA, 10, 7.

Publication 2019

PrimeStar HS BIOTaq CloneJET PCR Cloning Kit pGEM-T Easy Vector System Illustra plasmidPrep Mini Spin Kit

Agarose Dna sequences E coli Electrophoresis Electroporation Enzyme Gels Genome Nucleotide sequence Pgem Plasmids Primers Promega Vector

Corresponding Organization :

Other organizations : Universidad Publica de Navarra, Universidad de Málaga, Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora"

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Variable analysis

independent variables

Use of high fidelity enzyme (PrimeStar HS, Takara Bio Inc., Japan) or standard enzyme (BIOTaq, Bioline, UK) for DNA amplification

dependent variables

Amplicon cloning using CloneJET PCR Cloning Kit or pGEM-T Easy Vector System
Plasmid purification from E. coli using boiling method or commercial kit
Plasmid profile analysis by agarose gel electrophoresis
Plasmid transfer to P. syringae by electroporation
DNA sequence comparison and alignment using BLAST, MULTALIN, and EMBL-EBI tools
Protein motif search using InterPro interface
Nucleotide sequence visualization and manipulation using Artemis genome browser and ACT
Primer design using Primer3plus software

control variables

Primers detailed in Additional file 1 Table S1

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