Protocols in Current Issue
    Measuring Oligonucleotide Hydrolysis in Cellular Lysates via Viscosity Measurements
    Authors:  Romel Menacho-Melgar and Michael D. Lynch, date: 01/20/2022, view: 1127, Q&A: 0

    Cell lysis, a process that releases host oligonucleotides, is required in many biotechnological applications. However, intact oligonucleotides in crude cellular lysates increase the viscosity of lysates, which complicates downstream processes and routine laboratory workflows. To address this, nucleases that hydrolyze the intact oligonucleotides

    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 1894, Q&A: 0

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are difficult to model due to the helical character of the system. Here, we describe how distortions in RecA

    Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy
    Authors:  Yaqing Wang, Zhiqiang Sun, Piero R. Bianco and Yuri L. Lyubchenko, date: 03/05/2021, view: 3061, Q&A: 0

    In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks,

    Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids
    Authors:  Daniel C. Volke, Nicolas T. Wirth and Pablo I. Nikel, date: 02/20/2021, view: 3953, Q&A: 0

    Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ

    Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter
    Authors:  Yuki Yamanaka, Hiroki Watanabe, Erika Yamauchi, Yukari Miyake and Kaneyoshi Yamamoto, date: 01/20/2020, view: 3111, Q&A: 0
    [Abstract] The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced ...
    Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA
    Authors:  Martin Wegner, Koraljka Husnjak and Manuel Kaulich, date: 01/05/2020, view: 6268, Q&A: 0
    [Abstract] Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning ...
    High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach
    Authors:  Franziska M. Heydenreich, Tamara Miljuš, Dalibor Milić and Dmitry B. Veprintsev, date: 01/05/2020, view: 4013, Q&A: 0
    [Abstract] Site-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries ...
    Strand-specific Single-stranded DNA Sequencing (4S-seq) of E. coli genomes
    Authors:  Takahiro Masuda, Nobuaki Kono, Masaru Tomita and Kazuharu Arakawa, date: 08/05/2019, view: 5333, Q&A: 0
    [Abstract] Most bacterial genomes have biased nucleotide composition, and the asymmetry is considered to be caused by a single-stranded DNA (ssDNA) deamination arising from the bacterial replication machinery. In order to evaluate the relationship experimentally, the position and frequency of ssDNA formed during replication must be verified clearly. Although ...
    Bacterial Microcolonies in Gel Beads for High-throughput Screening
    Author:  Yolanda Schaerli, date: 07/05/2018, view: 7348, 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.
    Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
    Authors:  Prarthana Mohanraju, John van der Oost, Martin Jinek and Daan C. Swarts, date: 05/05/2018, view: 16701, Q&A: 3
    [Abstract] This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for ...

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