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

Authors: Gozde S. Demirer and Markita P. Landry, date: 01/05/2021, view: 409, 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 ...

Chromatin Immunoprecipitation (ChIP) to Assess Histone Marks in Auxin-treated Arabidopsis thaliana Inflorescence Tissue

Authors: André Kuhn and Lars Østergaard, date: 12/05/2020, view: 745, Q&A: 0

[Abstract] Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) or high-throughput sequencing (ChIP-seq) has become the gold standard for the identification of binding sites of DNA binding proteins and the localization of histone modification on a locus-specific or genome-wide scale, respectively. ChIP experiments can be divided into seven ...

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: 994, 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 ...

Ratiometric Measurement of Protein Abundance after Transient Expression of a Transgene in Nicotiana benthamiana

Authors: Aashima Khosla and David C. Nelson, date: 09/05/2020, view: 1752, Q&A: 0

[Abstract] Ratiometric reporters are tools to dynamically measure the relative abundance of a protein of interest. In these systems, a target protein fused to a fluorescent or bioluminescent reporter is expressed with fixed stoichiometry to a reference protein fused to a second reporter. Both fusion proteins are encoded on a single transcript but are ...

RNA Stability Measurements Using RT-qPCR in Arabidopsis Seedlings

Authors: Tianran Jia and Brandon H. Le, date: 07/20/2020, view: 890, Q&A: 0

[Abstract] Steady-state mRNA levels are determined by both the rates of transcription and degradation. Regulation of mRNA stability and/or degradation are key factors that can significantly affect mRNA levels and its biological functions. mRNA stability can be measured indirectly after transcription inhibition. This protocol described a rapid and sensitive ...

Methylation-sensitive Amplified Polymorphism as a Tool to Analyze Wild Potato Hybrids

Authors: Nicolás Cara, Carlos F. Marfil, María V. Bertoldi and Ricardo W. Masuelli, date: 07/05/2020, view: 1131, Q&A: 0

[Abstract] Methylation-Sensitive Amplification Polymorphism (MSAP) is a versatile marker for analyzing DNA methylation patterns in non-model species. The implementation of this technique does not require a reference genome and makes it possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome. In addition, ...

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

Authors: Susanna Keriö, Eeva Terhonen and Jared M. LeBoldus, date: 06/05/2020, view: 2200, 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 ...

High-resolution and Deep Phylogenetic Reconstruction of Ancestral States from Large Transcriptomic Data Sets

Authors: Sumanth Kumar Mutte and Dolf Weijers, date: 03/20/2020, view: 1789, Q&A: 0

[Abstract] Phylogenetics is an important area of evolutionary biology that helps to understand the origin and divergence of genes, genomes and species. Building meaningful phylogenetic trees is needed for the accurate reconstruction of the past. To achieve a correct phylogenetic understanding of genes or proteins, reliable and robust methods are needed to ...

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: 1759, 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 ...

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: 01/20/2020, view: 141, 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 ...

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