Molecular Biology

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    Protocols in Current Issue
    Attachment of a 32P-phosphate to the 3′ Terminus of a DNA Oligonucleotide
    Authors:  Joshua C. Cofsky and Jennifer A. Doudna, date: 10/20/2020, view: 211, Q&A: 0
    Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one ...
    A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP)
    SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all ...
    Analyzing (Re)Capping of mRNA Using Transcript Specific 5' End Sequencing
    Authors:  Daniel del Valle Morales and Daniel R. Schoenberg, date: 10/20/2020, view: 229, Q&A: 0
    The 5′ cap is a ubiquitous feature of eukaryotic mRNAs. It is added in the nucleus onto newly synthesized pre-mRNA, and in the cytoplasm onto mRNAs after decapping or endonuclease cleavage. Cytoplasmic recapping can occur after loss of the cap at ...
    Understanding Docking Complexes of Macromolecules Using HADDOCK: The Synergy between Experimental Data and Computations
    Authors:  Andrea Saponaro, Vincenzo Maione, Alexandre M. J. J. Bonvin and Francesca Cantini, date: 10/20/2020, view: 263, Q&A: 0
    This protocol illustrates the modelling of a protein-peptide complex using the synergic combination of in silico analysis and experimental results. To this end, we use the integrative modelling software HADDOCK, which possesses the powerful ...
    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: 298, Q&A: 0
    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 ...
    Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
    Author:  Marcelo S. da Silva, date: 10/20/2020, view: 296, Q&A: 0
    Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication ...
    Attachment of a 32P-phosphate to the 3′ Terminus of a DNA Oligonucleotide
    Authors:  Joshua C. Cofsky and Jennifer A. Doudna, date: 10/20/2020, view: 211, Q&A: 0
    [Abstract] Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one with the 5′ DNA end labeled and one with the 3′ DNA end labeled. While homogeneous 5′-radiolabeling ...
    A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP)
    [Abstract] SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown ...
    Analyzing (Re)Capping of mRNA Using Transcript Specific 5' End Sequencing
    Authors:  Daniel del Valle Morales and Daniel R. Schoenberg, date: 10/20/2020, view: 229, Q&A: 0
    [Abstract] The 5′ cap is a ubiquitous feature of eukaryotic mRNAs. It is added in the nucleus onto newly synthesized pre-mRNA, and in the cytoplasm onto mRNAs after decapping or endonuclease cleavage. Cytoplasmic recapping can occur after loss of the cap at the native 5′ end, or downstream within the body of the mRNA. The identification and location of ...
    Understanding Docking Complexes of Macromolecules Using HADDOCK: The Synergy between Experimental Data and Computations
    Authors:  Andrea Saponaro, Vincenzo Maione, Alexandre M. J. J. Bonvin and Francesca Cantini, date: 10/20/2020, view: 263, Q&A: 0
    [Abstract] This protocol illustrates the modelling of a protein-peptide complex using the synergic combination of in silico analysis and experimental results. To this end, we use the integrative modelling software HADDOCK, which possesses the powerful ability to incorporate experimental data, such as NMR Chemical Shift Perturbations and biochemical ...
    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: 298, 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 ...
    Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
    Author:  Marcelo S. da Silva, date: 10/20/2020, view: 296, Q&A: 0
    [Abstract] Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the ...
    TetR Regulated in vivo Repression Technology to Identify Conditional Gene Silencing in Genetically Engineerable Bacteria Using Vibrio cholerae Murine Infections as Model System
    Authors:  Franz G. Zingl, Fabian Mitterer, Himadri B. Thapa and Stefan Schild, date: 10/05/2020, view: 217, Q&A: 0
    [Abstract] Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to ...
    Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs
    Authors:  Megan D. Schertzer, McKenzie M. Murvin and J. Mauro Calabrese, date: 10/05/2020, view: 248, Q&A: 0
    [Abstract] Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA’s intracellular abundance, i.e., approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of ...
    Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses
    [Abstract] Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and ...
    Assembly and Imaging Set up of PIE-Scope
    [Abstract] Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy ...



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