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Preparation and Characterization of Internally Modified DNA Templates for Chemical Transcription Roadblocking

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Measurement of Transgene Copy Number in Plants Using Droplet Digital PCR

Authors: Yao-Min Cai, Quentin M. Dudley and Nicola J. Patron, date: 07/05/2021, view: 1291, Q&A: 0

[Abstract]

Transgenic plants are produced both to investigate gene function and to confer desirable traits into crops. Transgene copy number is known to influence expression levels, and consequently, phenotypes. Similarly, knowledge of transgene zygosity is desirable for making quantitative assessments of phenotype and tracking the inheritance of transgenes

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Cellulase and Macerozyme-PEG-mediated Transformation of Moss Protoplasts

Authors: Rituraj Batth, Ilenuta Simina Cuciurean, Satish Kumar Kodiripaka, Sarah Skovlund Rothman, Caroline Greisen and Henrik Toft Simonsen, date: 01/20/2021, view: 1610, Q&A: 0

[Abstract] This protocol describes the generation of protoplasts from protonemal tissue of the moss Physcomitrium patens (syn. Physcomitrella patens), using Cellulase ONOZUKA R10 and Macerozyme R10, followed by polyethylene glycol (PEG) mediated transformation. The protonemal tissue grown in liquid suspension was harvested and treated with ...

Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers

Authors: Gozde S. Demirer and Markita P. Landry, date: 01/05/2021, view: 2453, Q&A: 0

[Abstract] Gene knock-down in plants is a useful approach to study genotype-phenotype relationships, render disease resistance to crops, and enable efficient biosynthesis of molecules in plants. Small interfering RNA (siRNA)-mediated gene silencing is one of the most common ways to achieve gene knock-down in plants. Traditionally, siRNA is delivered into ...

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: 2711, 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: 3717, 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: 2823, 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: 5359, 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: 6126, 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: 6087, 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: 14013, 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 ...

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