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In vitro Protein-DNA Binding Assay (AlphaScreen^® Technology)

Authors: Mika Nomoto, Yasuomi Tada and Hironaka Tsukagoshi, date: 02/05/2019, view: 2151, Q&A: 0

[Abstract] Identification of specific DNA binding sites of transcription factors is important in understanding their functions. Recent techniques allow us to investigate genome-wide in vivo binding positions by chromatin immunoprecipitation combined with high-throughput sequencing. However, to further explore the binding motifs of transcription ...

Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly

Authors: Axel Himmelbach, Ines Walde, Martin Mascher and Nils Stein, date: 08/05/2018, view: 3071, Q&A: 0

[Abstract] Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic ...

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: 3071, 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 ...

An Optimized CTAB Method for Genomic DNA Extraction from Freshly-picked Pinnae of Fern, Adiantum capillus-veneris L.

Authors: Yi Shu, Jin Wan-Ting, Yuan Ya-Ning and Fang Yu-Han, date: 07/05/2018, view: 8557, Q&A: 0

[Abstract] As the sister clade of seed plants, ferns are significant materials for plant phylogeny research. However, the genomic DNA extraction protocol for fern samples like modified CTAB method still lacks robustness. Here, we found that the amount and condition of the pinnae samples are critical for gDNA extraction in fern, Adiantum capillus-veneris ...

Reduced Representation Bisulfite Sequencing in Maize

Authors: Fei-Man Hsu, Chung-Ju Rachel Wang and Pao-Yang Chen, date: 03/20/2018, view: 3453, Q&A: 0

[Abstract] DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 ...

Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis

Authors: Fantao Kong and Yonghua Li-Beisson, date: 02/05/2018, view: 3215, Q&A: 0

[Abstract] Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been ...

Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes

Authors: Moritz Bömer, Aliyu A. Turaki, Ajith I. Rathnayake, Gonçalo Silva, P. Lava Kumar and Susan E. Seal, date: 01/05/2018, view: 3823, Q&A: 0

[Abstract] Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem ...

Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USER^TM Cloning

Authors: Morten Egevang Jørgensen, Nikolai Wulff, Majse Nafisi, Deyang Xu, Cuiwei Wang, Sophie Konstanze Lambertz, Zeinu Mussa Belew and Hussam Hassan Nour-Eldin, date: 11/20/2017, view: 4749, Q&A: 0

[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 USER^TM 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: 6841, 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, ...

TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions

Authors: Amit K. Tripathi, Ashwani Pareek and Sneh Lata Singla-Pareek, date: 08/20/2017, view: 7306, Q&A: 0

[Abstract] DNA damage is one of the common consequences of exposure to various stress conditions. Different methods have been developed to accurately assess DNA damage and fragmentation in cells and tissues exposed to different stress agents. However, owing to the presence of firm cellulosic cell wall and phenolics, plant cells and tissues are not easily ...

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