Protocols in Current Issue
    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: 5701, 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 ...
    Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences
    Authors:  Rachael M. Giersch and Gregory C. Finnigan, date: 09/20/2017, view: 8846, Q&A: 0
    [Abstract] Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded ...
    Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System
    Authors:  Miriam Manor and Udi Qimron, date: 08/05/2017, view: 8138, Q&A: 0
    [Abstract] We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous ...
    Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae
    Authors:  Huanhuan Hao, Jing Huang, Tongtong Liu, Hui Tang and Liping Zhang, date: 07/20/2017, view: 9905, Q&A: 0
    [Abstract] CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very ...
    CRISPR/Cas9 Editing of the Bacillus subtilis Genome
    Authors:  Peter E. Burby and Lyle A. Simmons, date: 04/20/2017, view: 15227, Q&A: 4
    [Abstract] A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with ...
    Estimation of the Chromosomal Copy Number in Synechococcus elongatus PCC 7942
    Authors:  Satoru Watanabe and Hirofumi Yoshikawa, date: 07/05/2016, view: 6461, Q&A: 0
    [Abstract] Cyanobacteria are prokaryotic organisms that perform oxygenic photosynthesis. Freshwater cyanobacteria, such as Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, are model organisms for the study of photosynthesis, gene regulation, and biotechnological applications because they are easy to manipulate ...
    MNase Digestion for Nucleosome Mapping in Neurospora
    Authors:  Cigdem Sancar, Gencer Sancar and Michael Brunner, date: 06/05/2016, view: 7636, Q&A: 0
    [Abstract] Digestion of chromatin by micrococcal nuclease MNase followed by high throughput sequencing allows us to determine the location and occupancy of nucleosomes on the genome. Here in this protocol we have described optimized conditions of MNase digestion of filamentous fungus Neurospora crassa chromatin without a requirement of a nuclear ...
    Measuring UV-induced Mutagenesis at the CAN1 Locus in Saccharomyces cerevisiae
    Authors:  Ildiko Unk and Andreea Daraba, date: 10/20/2014, view: 9939, Q&A: 1
    [Abstract] There are several methods to measure the capacity of yeast cell to respond to environmental impacts on their genome by mutating it. One frequently used method involves the detection of forward mutations in the CAN1 gene. The CAN1 gene encodes for an arginine permease that is responsible for the uptake of arginine and it can also ...
    Chromatin Fractionation Assay in Fission Yeast
    Authors:  Tatsuki Kunoh and Toshiyuki Habu, date: 07/20/2014, view: 9748, Q&A: 0
    [Abstract] The protein recruitment onto chromatin is a critical process for DNA metabolism, including DNA replication, DNA repair and DNA recombination. Especially DNA modification enzymes and checkpoint proteins are loaded onto DNA damage sites in a context-dependent manner. In our recent study (Kunoh and Habu, 2014), the chromatin association of Pcf1, a ...
    Rhodobacter capsulatus Gene Transfer Agent Transduction Assay
    Authors:  Molly M. Leung and John Thomas Beatty, date: 02/20/2013, view: 8886, Q&A: 0
    [Abstract] The gene transfer agent (GTA) is a bacteriophage-like particle that transfers genomic DNA from a donor to a recipient bacterium. The Rhodobacter capsulatus GTA (RcGTA) was the first to be studied and this protocol has been optimized for RcGTA transduction, although it could be modified for other bacteria containing a GTA. The RcGTA ...

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