Molecular Biology

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    Protocols in Current Issue
    Antisense Oligo Pulldown of Circular RNA for Downstream Analysis
    Authors:  Debojyoti Das, Aniruddha Das and Amaresh C. Panda, date: 07/20/2021, view: 399, Q&A: 0

    Circular RNAs (circRNAs) are a large family of noncoding RNA molecules that have emerged as novel regulators of gene expression by sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs). Several computational tools have been developed to

    ...
    A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout in Various Vertebrate Cell Lines
    Authors:  Kohei Nishimura and Tatsuo Fukagawa, date: 07/20/2021, view: 360, Q&A: 0

    Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across

    ...
    A Gel-Based Assay for Probing Protein Translocation on dsDNA
    Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 982, Q&A: 0

    Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves

    ...
    A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells
    Authors:  Amanda L. Koch, Tatsuya Morisaki and Timothy J. Stasevich, date: 07/20/2021, view: 812, Q&A: 0

    Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This

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    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 977, Q&A: 0

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are

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    GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish

    Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for

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    GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish
    [Abstract]

    Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging)

    ...
    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 977, Q&A: 0
    [Abstract]

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are difficult to model due to the helical character of the system. Here, we describe how distortions in RecA

    ...
    A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells
    Authors:  Amanda L. Koch, Tatsuya Morisaki and Timothy J. Stasevich, date: 07/20/2021, view: 812, Q&A: 0
    [Abstract]

    Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This technique employs a pair of complementary intrabodies loaded into living cells that co-translationally bind

    ...
    A Gel-Based Assay for Probing Protein Translocation on dsDNA
    Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 982, Q&A: 0
    [Abstract]

    Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves multiple DNA binding states, which are usually dependent on nucleotide binding/hydrolysis and are

    ...
    A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout in Various Vertebrate Cell Lines
    Authors:  Kohei Nishimura and Tatsuo Fukagawa, date: 07/20/2021, view: 360, Q&A: 0
    [Abstract]

    Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across a variety of eukaryotes; however, this type of simple gene knockout strategy is not applicable to

    ...
    Antisense Oligo Pulldown of Circular RNA for Downstream Analysis
    Authors:  Debojyoti Das, Aniruddha Das and Amaresh C. Panda, date: 07/20/2021, view: 399, Q&A: 0
    [Abstract]

    Circular RNAs (circRNAs) are a large family of noncoding RNA molecules that have emerged as novel regulators of gene expression by sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs). Several computational tools have been developed to predict circRNA interaction with target miRNAs and RBPs with a view to studying their potential effect

    ...
    PCR-mediated One-day Synthesis of Guide RNA for the CRISPR/Cas9 System
    Authors:  Naim Hassan, Farhana Easmin, Keisuke Ekino and Satoshi Harashima, date: 07/05/2021, view: 1133, Q&A: 0
    [Abstract]

    Nowadays, CRISPR (clustered regularly interspaced short palindromic repeats) and the CRISPR-associated protein (Cas9) system play a major role in genome editing. To target the desired sequence of the genome successfully, guide RNA (gRNA) is indispensable for the CRISPR/Cas9 system. To express gRNA, a plasmid expressing the gRNA sequence is

    ...
    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: 1009, 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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    Transcriptional Run-on: Measuring Nascent Transcription at Specific Genomic Sites in Yeast
    Authors:  Victoria Begley, Lola de Miguel-Jiménez and Sebastián Chávez, date: 06/20/2021, view: 758, Q&A: 0
    [Abstract]

    DNA transcription by RNA polymerases has always interested the scientific community as it is one of the most important processes involved in genome expression. This has led scientists to come up with different protocols allowing analysis of this process in specific locations across the genome by quantitating the amount of RNA polymerases

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    Peptide-mediated Targeting of Nanoparticles with Chemical Cargoes to Chloroplasts in Arabidopsis Plants
    Authors:  Israel Santana, Peiguang Hu, Su-Ji Jeon, Chris Castillo, Hann Tu and Juan Pablo Giraldo, date: 06/20/2021, view: 987, Q&A: 0
    [Abstract]

    Plant nanobiotechnology is a flourishing field that uses nanomaterials to study and engineer plant function. Applications of nanotechnology in plants have great potential as tools for improving crop yield, tolerance to disease and environmental stress, agrochemical delivery of pesticides and fertilizers, and genetic modification and transformation

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