Protocols in Current Issue
    Investigation of Transposon DNA Methylation and Copy Number Variation in Plants Using Southern Hybridisation
    Authors:  Vivek Hari Sundar G. and P. V. Shivaprasad, date: 06/05/2022, view: 873, Q&A: 0

    Plant genomes are pronouncedly enriched in repeat elements such as transposons. These repeats are epigenetically regulated by DNA methylation. Whole genome high-depth sequencing after bisulfite treatment remains an expensive and laborious method to reliably profile the DNA methylome, especially when considering large genomes such as in crops.

    Identification and Monitoring of Nucleotide Repeat Expansions Using Southern Blotting in Drosophila Models of C9orf72 Motor Neuron Disease and Frontotemporal Dementia
    Authors:  Joanne L. Sharpe, Nikki S. Harper and Ryan J. H. West, date: 05/20/2022, view: 710, Q&A: 0

    Repeat expansion diseases, including fragile X syndrome, Huntington’s disease, and C9orf72-related motor neuron disease and frontotemporal dementia, are a group of disorders associated with polymorphic expansions of tandem repeat nucleotide sequences. These expansions are highly repetitive and often hundreds to thousands of repeats in

    ATAC Sequencing Protocol For Cryopreserved Mammalian Cells
    Authors:  Juan Manuel Caravaca, Monika Mehta, Sujatha Gowda and Bao Tran, date: 01/20/2022, view: 2067, Q&A: 0

    ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) is a powerful method to evaluate chromatin accessibility and nucleosome positioning at a genome-wide scale. This assay uses a hyperactive Tn5 transposase, to simultaneously cut open chromatin and insert adapter sequences. After sequencing, the reads generated

    Single Molecular Resolution to Monitor DNA Replication Fork Dynamics upon Stress by DNA Fiber Assay
    Author:  Wenpeng Liu, date: 12/20/2021, view: 1653, Q&A: 0

    DNA replication always encounters numerous endogenous and exogenous stresses during the cell cycle. Measuring the cell responses to stress has primarily relied on cell survival and incorporation of radioactive dNTPs, which is limited in resolution. A higher resolution is required to monitor how replication and repair respond to these stresses.

    Reconstitution of Chromatin by Stepwise Salt Dialysis
    Authors:  Grisel Cruz-Becerra and James T. Kadonaga, date: 04/05/2021, view: 2961, Q&A: 0

    Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields chromatin that consists of purified histones and DNA. This biochemically pure chromatin is well-suited for

    Assembly of Genetic Circuits with the Mammalian ToolKit
    Authors:  João P. Fonseca, Alain R Bonny, Jason Town and Hana El-Samad, date: 03/05/2020, view: 4374, Q&A: 0
    [Abstract] The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. The Mammalian ToolKit (MTK) is a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of ...
    Construction of Antisense RNA-mediated Gene Knock-down Strains in the Cyanobacterium Anabaena sp. PCC 7120
    Authors:  Amit Srivastava, Anand Ballal, Karl Forchhammer and Anil Kumar Tripathi, date: 02/20/2020, view: 4012, Q&A: 0
    [Abstract] Anabaena sp. PCC 7120 (hereafter Anabaena) is a model cyanobacterium to study nitrogen fixation, cellular differentiation and several other key biological functions that are analogous in plants. As with any other organism, many genes in Anabaena encode an essential life function and hence cannot be deleted, causing a ...
    Evaluation of Genotoxicity by Micronucleus Assay in vitro and by Allium cepa Test in vivo
    Authors:  Christina N. Banti and Sotiris K. Hadjikakou, date: 07/20/2019, view: 6788, Q&A: 0
    [Abstract] The in vitro and in vivo genotoxicity of new metallodrugs either as Small Bioactive Molecules (SBAMs) or Conjugates of Metals with Drugs (CoMeDs) is evaluated by the micronucleus test and the Allium cepa assay, respectively. Fetal lung fibroblast cells (MRC-5), normal human corneal epithelial cells (HCEC) and ...
    DNA Fiber Assay upon Treatment with Ultraviolet Radiations
    Authors:  Alfano Luigi, Antonio Giordano and Francesca Pentimalli, date: 06/05/2017, view: 17871, Q&A: 1
    [Abstract] Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well ...
    Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
    Authors:  Hélène Tourrière, Julie Saksouk, Armelle Lengronne and Philippe Pasero , date: 06/05/2017, view: 12458, Q&A: 0
    [Abstract] The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical ...

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