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Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis

Authors: Yu Jin and Sebastian Marquardt, date: 10/20/2020, view: 146, Q&A: 0

[Abstract] CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an ...

Safe DNA-extraction Protocol Suitable for Studying Tree-fungus Interactions

Authors: Susanna Keriö, Eeva Terhonen and Jared M. LeBoldus, date: 06/05/2020, view: 1631, Q&A: 0

[Abstract] We present a safe and low-cost method suitable for DNA extraction from mycelium and tree tissue samples. After sample preparation, the extraction takes about 60 min. Method performance was tested by extracting DNA from various tree tissue samples and from mycelium grown on solid and liquid media. DNA was extracted from juvenile and mature host ...

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: 1462, 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 ...

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

Authors: Mika Nomoto, Yasuomi Tada and Hironaka Tsukagoshi, date: 02/05/2019, view: 3790, 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: 4629, 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: 4626, 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: 11812, 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: 4638, 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: 4432, 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: 5488, 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 ...

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