Protocols in Current Issue
    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: 671, 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: 1315, Q&A: 0
    [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 ...
    Root Aliphatic Suberin Analysis Using Non-extraction or Solvent-extraction Methods
    Authors:  Camille Delude, Sollapura J. Vishwanath, Owen Rowland and Frédéric Domergue, date: 06/20/2017, view: 795, Q&A: 0
    [Abstract] Here we describe both non-extraction and solvent-extraction methods for root aliphatic suberin analysis. The non-extraction method is fast as roots are directly depolymerized using acidic transmethylation. However, suberin aliphatic components are isolated together with all the other acyl chains making up the lipids (e.g., membranes) ...
    Simple Spectroscopic Determination of Nitrate, Nitrite, and Ammonium in Arabidopsis thaliana
    Authors:  Takushi Hachiya and Yuki Okamoto, date: 05/20/2017, view: 1299, Q&A: 1
    [Abstract] Plants use nitrate, nitrite, and ammonium as inorganic nitrogen (N) sources. These N compounds are included in plant tissues at various concentrations depending on the balance between their uptake and assimilation. Thus, the contents of nitrate, nitrite, and ammonium are physiological indicators of plant N economy. Here, we describe a protocol for ...
    Mating Based Split-ubiquitin Assay for Detection of Protein Interactions
    Authors:  Wijitra Horaruang and Ben Zhang, date: 05/05/2017, view: 1417, Q&A: 0
    [Abstract] The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are ...
    Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography
    Authors:  Valentina La Verde, Paola Dominici and Alessandra Astegno, date: 04/20/2017, view: 1703, Q&A: 0
    [Abstract] Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties ( ...
    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: 1697, 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: 2434, 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 ...
    Nitrate Assay for Plant Tissues
    Authors:  Lufei Zhao and Yong Wang, date: 01/20/2017, view: 2333, Q&A: 0
    [Abstract] Nitrogen is an essential macronutrient for plant growth and nitrate content in plants can reflect the nitrogen supply of soil. Here, we provide the salicylic acid method to evaluate the nitrate content in plant tissues. The method is reliable and stable, thus it can be a good choice for measurement of nitrate in plant tissues.
    Pseudomonas syringae Flood-inoculation Method in Arabidopsis
    Authors:  Yasuhiro Ishiga, Takako Ishiga, Yuki Ichinose and Kirankumar S. Mysore, date: 01/20/2017, view: 1627, Q&A: 0
    [Abstract] Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen because of its pathogenicity on Arabidopsis thaliana. Here, we demonstrate a rapid and reliable flood-inoculation method based on young Arabidopsis seedlings ...