Protocols in Current Issue
    PCR-mediated One-day Synthesis of Guide RNA for the CRISPR/Cas9 System
    Authors:  Naim Hassan, Farhana Easmin, Keisuke Ekino and Satoshi Harashima, date: 07/05/2021, view: 1118, Q&A: 0

    Nowadays, CRISPR (clustered regularly interspaced short palindromic repeats) and the CRISPR-associated protein (Cas9) system play a major role in genome editing. To target the desired sequence of the genome successfully, guide RNA (gRNA) is indispensable for the CRISPR/Cas9 system. To express gRNA, a plasmid expressing the gRNA sequence is

    Transcriptional Run-on: Measuring Nascent Transcription at Specific Genomic Sites in Yeast
    Authors:  Victoria Begley, Lola de Miguel-Jiménez and Sebastián Chávez, date: 06/20/2021, view: 752, Q&A: 0

    DNA transcription by RNA polymerases has always interested the scientific community as it is one of the most important processes involved in genome expression. This has led scientists to come up with different protocols allowing analysis of this process in specific locations across the genome by quantitating the amount of RNA polymerases

    Defined Mutant Library Sequencing (DML-Seq) for Identification of Conditional Essential Genes
    Authors:  Shuai Shao, Lifan Wei, Feng Xia, Yuanxing Zhang and Qiyao Wang, date: 03/05/2021, view: 1756, Q&A: 0

    Transposon insertion sequencing (TIS) is an emerging technique which utilizes a massive transposon mutant library to screen specific phenotype and determine the conditional essential genetic requirements for bacterial fitness under distinct conditions combined with high-throughput parallel sequencing technology. Compared with a massive mutant

    Bacterial Conjugation Protocol for Ruminant Mycoplasmas
    Authors:  Eveline Sagné, Christine Citti and Emilie Dordet-Frisoni, date: 01/20/2021, view: 846, Q&A: 0

    In Mycoplasma agalactiae, two simultaneous processes of DNA transfer have been described that require direct cell-to-cell contact and are similar to conjugation. One involves the self-transmission of an integrative conjugative element (ICE) while the second concerns the horizontal transfer of large and small fragments of chromosomal DNA. Here, we

    Candida albicans Culture, Cell Harvesting, and Total RNA Extraction
    Authors:  Max V. Cravener and Aaron P. Mitchell, date: 11/05/2020, view: 1340, Q&A: 0

    Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the

    Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
    Author:  Marcelo S. da Silva, date: 10/20/2020, view: 1457, Q&A: 0
    [Abstract] Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the ...
    TetR Regulated in vivo Repression Technology to Identify Conditional Gene Silencing in Genetically Engineerable Bacteria Using Vibrio cholerae Murine Infections as Model System
    Authors:  Franz G. Zingl, Fabian Mitterer, Himadri B. Thapa and Stefan Schild, date: 10/05/2020, view: 997, Q&A: 0
    [Abstract] Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to ...
    Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria
    Authors:  Ramón Cervantes-Rivera and Andrea Puhar, date: 09/20/2020, view: 2688, Q&A: 0
    [Abstract] Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene ...
    Efficient Agrobacterium-mediated Transformation of the Elite–Indica Rice Variety Komboka
    [Abstract] Genetic transformation is crucial for both investigating gene functions and for engineering of crops to introduce new traits. Rice (Oryza sativa L.) is an important model in plant research, since it is the staple food for more than half of the world’s population. As a result, numerous transformation methods have been developed for both ...
    Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9
    Authors:  Gloria Muñoz-Fernández, Alberto Jiménez and José Luis Revuelta, date: 06/20/2020, view: 1677, Q&A: 0
    [Abstract] The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus A. gossypii is currently used for the industrial production of riboflavin (vitamina B2). In ...

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