Current Protocols in Molecular Biology最新文献

We describe the procedure for inoculating overnight (starter) cultures of E. coli from a single colony, along with considerations for growing larger cultures. We also include two methods for monitoring the number of cells per unit volume (density) of liquid cultures using a spectrophotometer and a hemacytometer or “count slide.” © 2018 by John Wiley & Sons, Inc.

In the past few decades, numerous approaches have been developed to investigate protein-protein and protein-nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co-immunoprecipitation (co-IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two-step co-immunoprecipitation (TIP) technique. As compared to standard co-IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein-protein complex from Burkitt lymphoma cells and from primary human CD4⁺ T cells. In addition, this unit describes an application of TIP for the isolation of transcription-factor-bound chromatin. © 2018 by John Wiley & Sons, Inc.

Differential Scanning Fluorimetry Guided Refolding (DGR) is a simple methodology that can be used to rapidly screen for and identify conditions capable of accurately refolding protein preparations, such as those obtained from Escherichia coli inclusion bodies. It allows for the production in E. coli of functional proteins that would otherwise require far more expensive production methods. This unit describes how to set up a DGR refolding assay, perform DGR refolding trials in microplate format, use MeltTraceur Web software to interactively analyze the resulting data, scale-up protein production via refolding, and lastly, validate that the protein is properly folded. © 2018 by John Wiley & Sons, Inc.

Post-transcriptional regulation is an important aspect of the control of gene expression. mRNAs are translated with variable efficiencies, and these efficiencies can change rapidly during adaptation to diverse environmental factors, including cellular stresses and microbial infections. Polysome profiling analysis utilizes ultracentrifugation to isolate complexes of mRNAs in the process of translation and corresponding proteins on the basis of density. Here we describe polysome profiling analysis using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR analysis and isolation of protein for SDS-PAGE and immunoblot analysis. © 2018 by John Wiley & Sons, Inc.

This unit describes a reverse transcription-quantitative PCR (RT-qPCR)–based method for gene-targeted measurement of RNA translation levels. The method includes washing and lysing cells with a buffer containing cycloheximide to enrich ribosomal accumulation at translation initiation sites (TIS), followed by enzymatic treatment to generate ribosomal footprints, reverse transcription targeted towards TIS of specific transcripts of interest to generate complementary DNA (cDNA), and qPCR to measure the abundance of these footprints. This method enables time- and cost-effective assessment of changes in translation levels across focused panels of genes and across numerous samples. © 2018 by John Wiley & Sons, Inc.

Identification of spontaneous or chemically induced bacterial mutations is a powerful tool for investigation of molecular mechanisms, including the mechanism of action of novel antibiotics. However, a major bottleneck to this approach has been the identification of the causative mutation underlying a phenotype of interest. Until recently, this has required time-consuming genetic analysis. However, the advent of relatively inexpensive and rapid next-generation sequencing (NGS) technologies has revolutionized the correlation of bacterial phenotypes and genotypes. In this article we describe a simple bioinformatics pipeline to identify differences between sequenced bacterial genomes. We also describe the procedures involved in growing, extracting, and purifying DNA, and preparation of sequencing libraries for one bacterial species, Pseudomonas aeruginosa. Similar protocols will be applicable to other bacterial species. © 2018 by John Wiley & Sons, Inc.

Quantitative analysis of gene expression is crucial for understanding the molecular mechanisms underlying genome regulation. RNA-seq is a powerful platform for comprehensive investigation of the transcriptome. In this unit, we present a general bioinformatics workflow for the quantitative analysis of RNA-seq data and describe a few current publicly available computational tools applicable at various steps of this workflow. These tools comprise a pipeline for quality assessment and quantitation of RNA-seq data that starts from raw sequencing files and is focused on the identification and analysis of genes that are differentially expressed between biological conditions. © 2018 by John Wiley & Sons, Inc.

RNAi is a powerful reverse genetics tool that has revolutionized genetic studies in model organisms. The bacteriovorous nematode Caenorhabditis elegans can be genetically manipulated by feeding it an Escherichia coli strain that expresses a double-stranded RNA (dsRNA) corresponding to a C. elegans gene, which leads to systemic silencing of the gene. This unit describes protocols for performing an automated high-throughput RNAi screen utilizing a full-genome C. elegans RNAi library. The protocols employ liquid-handling robotics and 96-well plates. © 2018 by John Wiley & Sons, Inc.

Automated or semi-automated high-throughput RNAi screens are highly prone to systematic errors because of multistep repetitive protocols and extensive use of automated instruments. A well-designed RNAi library can help detect and minimize systematic human and robotic errors. In this unit, we describe how to design an RNAi bacterial library for use in conjunction with the well-studied nematode Caenorhabditis elegans for automated phenotypic screens. We provide strategies to design and assemble RNAi libraries to reduce or eliminate systematic errors. These strategies serve as a good quality-control check and facilitate obtaining high-quality data from a genome-wide and sub-library RNAi screen. © 2018 by John Wiley & Sons, Inc.