Protocols in Current Issue
    Improved HTGTS for CRISPR/Cas9 Off-target Detection
    Authors:  Jianhang Yin, Mengzhu Liu, Yang Liu and Jiazhi Hu, date: 05/05/2019, view: 260, Q&A: 0
    [Abstract] Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a “bait” DNA ...
    Precision Tagging: A Novel Seamless Protein Tagging by Combinational Use of Type II and Type IIS Restriction Endonucleases
    Authors:  Zhen Xu, Yan-Ning Rui, John P. Hagan and Dong H. Kim, date: 02/05/2018, view: 2867, Q&A: 0
    [Abstract] Protein tagging is a powerful tool for performing comprehensive analyses of the biological functions of a protein of interest owing to the existence of a wide variety of tags. It becomes indispensable in some cases, such as in tracking protein dynamics in a live cell or adding a peptide epitope due to the lack of optimal antibodies. However, ...
    NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells
    Authors:  Alvaro E. Galvis, Hugh E. Fisher and David Camerini, date: 10/20/2017, view: 5214, Q&A: 3
    [Abstract] This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from ...
    Immunostaining of Formaldehyde-fixed Metaphase Chromosome from Untreated and Aphidicolin-treated DT40 Cells
    Author:  Vibe H. Oestergaard, date: 05/05/2017, view: 4632, Q&A: 0
    [Abstract] During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.

    This protocol describes a method to perform immunostaining of formaldehyde-fixed metaphase chromosomes from the avian cell line DT40. It was developed to ...
    Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells
    Authors:  Christin Herrmann, Daphne C. Avgousti and Matthew D. Weitzman, date: 03/20/2017, view: 7602, Q&A: 0
    [Abstract] Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome ...
    Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation
    [Abstract] dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start ...
    Karyotype Analysis
    Authors:  Chun-Hung Chou and Muh-Hwa Yang, date: 05/20/2014, view: 15043, Q&A: 0
    [Abstract] A chromosome is the structure that organizes DNA and protein in cells. It is a single piece of coiled DNA containing coding and non-coding sequences. Human cells have 23 pairs of chromosomes including 22 pairs of autosomes and one pair of sex chromosome, giving a total of 46 per cell. In tumor cells, chromosomal instability has been considered to ...
    Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP)
    Authors:  Toshitsugu Fujita and Hodaka Fujii, date: 05/20/2014, view: 8570, Q&A: 0
    [Abstract] Elucidation of molecular mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. To this end, it is useful to isolate the target regions retaining molecular interactions. We established locus-specific chromatin immunoprecipitation (ChIP) technologies consisting of ...
    5-Hydroxymethylcytosine (5-hmC) Specific Enrichment
    Authors:  Keith E Szulwach, Chun-Xiao Song, Chuan He and Peng Jin, date: 08/05/2012, view: 7018, Q&A: 0
    [Abstract] 5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA modification in mammalian genomes. This protocol is to be a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically, which could in turn be used for high-throughput mapping via ...
    Propidium Iodide Staining of Cells for FACS Analysis
    Author:  Hui Zhu, date: 06/05/2012, view: 36591, Q&A: 2
    [Abstract] Fluorescence Activated Cell Sorting (FACS) is used to study DNA cell content. Propidium iodide (PI) intercalates into double-stranded nucleic acids and fluoresces. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. Thus PI staining is included in immunofluorescent staining protocols to identify dead cells. DNA ...

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