Protocols in Current Issue
    A Molecular Cloning and Sanger Sequencing-based Protocol for Detecting Site-specific DNA Methylation
    Authors:  Wei Guo, Anthony Cannon and Damon Lisch, date: 05/05/2022, view: 489, Q&A: 0

    DNA methylation is a conserved chemical modification, by which methyl groups are added to the cytosine of DNA molecules. Methylation can influence gene expression without changing the sequence of a particular gene. This epigenetic effect is an intriguing phenomenon that has puzzled biologists for years. By probing the temporal and spatial patterns

    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: 6974, 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 ...
    Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USERTM Cloning
    [Abstract] This protocol describes how to order and directly assemble uracil-containing non-clonal DNA fragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal of making synthesized non-clonal DNA fragments directly compatible with USERTM cloning. The protocol is highly efficient and would be compatible ...
    Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants
    Authors:  Xu Tang, Zhaohui Zhong, Xuelian Zheng and Yong Zhang, date: 09/05/2017, view: 11389, Q&A: 0
    [Abstract] The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, ...
    Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9
    Authors:  Florian Hahn, Marion Eisenhut, Otho Mantegazza and Andreas P. M. Weber, date: 07/05/2017, view: 21337, Q&A: 1
    [Abstract] The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing ...
    Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9
    Authors:  Julia Schumacher, Kerstin Kaufmann and Wenhao Yan, date: 03/05/2017, view: 10514, Q&A: 0
    [Abstract] Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional ...
    Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters
    Authors:  Tao Zhang, Yangbin Gao, Rongchen Wang and Yunde Zhao, date: 02/20/2017, view: 15157, Q&A: 0
    [Abstract] CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct ...
    Chromosome Dosage Analysis in Plants Using Whole Genome Sequencing
    Authors:  Ek Han Tan, Luca Comai and Isabelle M. Henry, date: 07/05/2016, view: 10576, Q&A: 0
    [Abstract] Relative chromosome dosage, i.e., increases or decreases in the number of copies of specific chromosome regions in one sample versus another, can be determined using aligned read-counts from Illumina sequencing (Henry et al., 2010). The following protocol was used to identify the different classes of aneuploids that ...
    Analysis of Telomeric G-overhangs by in-Gel Hybridization
    Authors:  Sona Valuchova, Elisa Derboven and Karel Riha, date: 04/05/2016, view: 7429, Q&A: 0
    [Abstract] Telomeric DNA in majority of eukaryotes consists of an array of TG-rich tandem repeats. The TG-rich DNA strand is oriented with its 3’ end towards chromosome termini and is usually longer than its complementary CA-rich strand, thus forming 3’ single stranded overhang (G-overhang). G-overhangs arise from incomplete replication of chromosome termini ...
    Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths
    Authors:  Miloslava Fojtová, Petr Fajkus, Pavla Polanská and Jiří Fajkus, date: 12/05/2015, view: 15815, Q&A: 0
    [Abstract] Chromosome ends - telomeres - are a focus of intensive research due to their importance for the maintenance of chromosome stability. Their shortening due to incomplete replication functions as a molecular clock counting the number of cell divisions, and ultimately results in cell-cycle arrest and cellular senescence. Determination of telomere ...

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