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    Protocols in Current Issue
    Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USERTM Cloning
    [Abstract] This protocol describes how to order and directly assemble uracil-containing non-clonal DNA fragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal of making synthesized non-clonal DNA fragments directly compatible with USERTM cloning. The protocol is highly efficient and would be compatible ...
    Generation and Selection of Transgenic Olive Plants
    Authors:  Elena Palomo-Ríos, Sergio Cerezo, Jose Ángel Mercado and Fernando Pliego-Alfaro, date: 11/20/2017, view: 49, Q&A: 0
    [Abstract] Olive (Olea europaea L.) is one of the most important oil crops in the Mediterranean basin. Biotechnological improvement of this species is hampered by the recalcitrant nature of olive tissue to regenerate in vitro. In previous investigations, our group has developed a reliable Agrobacterium-mediated transformation ...
    In vitro Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (in vitro enChIP) Using CRISPR Ribonucleoproteins in Combination with Next-generation Sequencing (in vitro enChIP-Seq) for the Identification of Chromosomal Interactions
    Authors:  Toshitsugu Fujita and Hodaka Fujii, date: 11/20/2017, view: 51, Q&A: 0
    [Abstract] We have developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP). Locus-specific ChIP is a method to isolate a genomic region of interest from cells while it also identifies what binds to this region using mass ...
    Markerless Gene Editing in the Hyperthermophilic Archaeon Thermococcus kodakarensis
    Authors:  Alexandra M. Gehring, Travis J. Sanders and Thomas J. Santangelo, date: 11/20/2017, view: 64, Q&A: 0
    [Abstract] The advent of single cell genomics and the continued use of metagenomic profiling in diverse environments has exponentially increased the known diversity of life. The recovered and assembled genomes predict physiology, consortium interactions and gene function, but experimental validation of metabolisms and molecular pathways requires more ...
    Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles
    Authors:  Rubul Mout and Vincent M. Rotello, date: 10/20/2017, view: 735, Q&A: 0
    [Abstract] In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in ...
    NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells
    Authors:  Alvaro E. Galvis, Hugh E. Fisher and David Camerini, date: 10/20/2017, view: 620, Q&A: 1
    [Abstract] This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from ...
    Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli
    Authors:  Xin-tian Li, Cindy Sou and Suckjoon Jun, date: 10/05/2017, view: 770, Q&A: 0
    [Abstract] We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and ...
    Quantification of Densities of Bacterial Endosymbionts of Insects by Real-time PCR
    Author:  Daisuke Kageyama, date: 10/05/2017, view: 430, Q&A: 0
    [Abstract] Increased attention has been paid to the endosymbiotic bacteria of insects. Because most insect endosymbionts are uncultivable, quantitative PCR (qPCR) is a practical and convenient method to quantify endosymbiont titers. Here we report a protocol for real-time qPCR based on SYBR Green I fluorescence as well as some tips to prevent possible ...
    Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection
    Authors:  Matthias Rieckher and Nektarios Tavernarakis, date: 10/05/2017, view: 470, Q&A: 0
    [Abstract] Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is ...
    Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells
    Authors:  Dheeraj Singh Rathore, Fiona M. Doohan and Ewen Mullins, date: 10/05/2017, view: 473, Q&A: 0
    [Abstract] Ensifer adhaerens OV14, a soil borne alpha-proteobacteria of the Rhizobiaceae family, fortifies the novel plant transformation technology platform termed ‘Ensifer-mediated transformation’ (EMT). EMT can stably transform both monocot and dicot species, and the host range of EMT is continuously expanding across a diverse range of ...