Molecular Biology

Categories

    Protocols in Current Issue
    In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells
    [Abstract] The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating ...
    Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap
    Authors:  Jonas Mechtersheimer, Stefan Reber and Marc-David Ruepp, date: 10/20/2018, view: 751, Q&A: 0
    [Abstract] This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any ...
    Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor
    Authors:  Helen Wang, Samuel S. Slattery, Bogumil J. Karas and David R. Edgell, date: 08/20/2018, view: 858, Q&A: 0
    [Abstract] Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. Phaeodactylum tricornutum is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of ...
    Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone
    Authors:  Jeffrey W. Perry and Andrew W. Tai, date: 08/20/2018, view: 587, Q&A: 0
    [Abstract] Protein tagging is a powerful method of investigating protein function. However, modifying positive-strand RNA virus proteins in the context of viral infection can be particularly difficult as their compact genomes and multifunctional proteins mean even small changes can inactivate or attenuate the virus. Although targeted approaches to ...
    CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus
    [Abstract] Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of ...
    High Resolution Melting Temperature Analysis to Identify CRISPR/Cas9 Mutants from Arabidopsis
    Authors:  Cynthia Denbow, Sonia Carole Ehivet and Sakiko Okumoto, date: 07/20/2018, view: 916, Q&A: 0
    [Abstract] CRISPR/Cas9 made targeted mutagenesis and genome editing possible for many plant species. One of the ways that the endonuclease is used for plant genetics is the creation of loss-of-function mutants, which typically result from erroneous DNA repair through non-homologous end joining (NHEJ) pathway. The majority of erroneous repair events results ...
    Bacterial Microcolonies in Gel Beads for High-throughput Screening
    Author:  Yolanda Schaerli, date: 07/05/2018, view: 1872, Q&A: 0
    [Abstract] High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants.
    ...
    Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts
    Authors:  Andrew M. Tidball, Preethi Swaminathan, Louis T. Dang and Jack M. Parent, date: 04/05/2018, view: 2837, Q&A: 1
    [Abstract] For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector ...
    Synthetic Genetic Interaction (CRISPR-SGI) Profiling in Caenorhabditis elegans
    Authors:  John A. Calarco and Adam D. Norris, date: 03/05/2018, view: 1819, Q&A: 0
    [Abstract] Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and E. coli (Schuldiner et al., 2005; Butland et al., 2008; Costanzo et al., 2010), ...
    Assaying Thermo-nociceptive Behavior in Drosophila Larvae
    Authors:  Meike Petersen, Federico Tenedini, Nina Hoyer, Fritz Kutschera and Peter Soba, date: 02/20/2018, view: 1788, Q&A: 0
    [Abstract] Thermo-nociception, the detection and behavioral response to noxious temperatures, is a highly conserved action to avoid injury and ensure survival. Basic molecular mechanisms of thermal responses have been elucidated in several model organisms and are of clinical relevance as thermal hypersensitivity (thermos-allodynia) is common in neuropathic ...



    We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.