Current Protocols Essential Laboratory Techniques最新文献

This unit provides a step-by-step guide to help you turn your high-quality data into a high-quality manuscript for publication in a scientific journal. It covers all aspects of the writing process, including: choosing a journal to which to submit your paper, how to proceed through writing each section, formulating your “story,” making figures, soliciting constructive criticism, and navigating the review process. Curr. Protoc. Essential Lab. Tech. 2:A.5C.1-A.5C.18. © 2009 by John Wiley & Sons, Inc.

Proteomics is the study of the proteins expressed from the genome of a cell or organism. Analytical mass spectrometry has recently become a powerful tool to study a cell or organism's proteome and is the focus of this chapter. Mass spectrometry can be used for both qualitative and quantitative analysis of proteomes. This chapter will focus on qualitative analysis, addressing questions of what proteins are present in a proteome and what post-translational modifications may be associated with these proteins. The instrumentation required for mass spectrometric analysis is generally not available in a standard research laboratory or as part of an undergraduate laboratory, being associated in general with a core facility. We will not describe here details of specific operation of the instruments. Here we focus on the common types of analysis presently in routine use and on preparation of samples for routine biological mass spectrometric analysis that will allow most laboratories, including undergraduate and graduate teaching laboratories, to prepare and analyze samples in experiments designed to obtain proteomic information. Curr. Protoc. Essential Lab. Tech. 2:12.2.1-12.1.18. © 2009 by John Wiley & Sons, Inc.

Model Organism Databases (MODs) represent the union of database technology and biology, and are essential to modern biological and medical research. Research communities are producing floods of new data, of increasingly different types and complexity. MODs assimilate this information from a wide variety of sources, organize it in a comprehensible manner, and make it freely available to the public via the Internet. MODs permit researchers to sort through massive amounts of data, providing access to key information that they might otherwise have overlooked. The protocols in this unit offer a general introduction to different types of data available in the growing number of MODs, and approaches for accessing, browsing, and querying these data. Curr. Protoc. Essential Lab. Tech. 1:11.4.1-11.4.17. © 2009 by John Wiley & Sons, Inc.

BLAST is the most widely used software in bioinformatics research. Its main function is to compare a sequence of interest, the query sequence, to sequences in a large database. BLAST then reports the best matches, or “hits,” found in the database. This simple program has two primary applications. First, if the function of the query sequence is unknown, it may be possible to infer its function based on the recognized functions of similar sequences. Second, if the researcher has a query sequence with a known function, it may be possible to identify sequences in the database that have similar functions. The utility of BLAST therefore depends on the researcher's choice of query sequence and database. An appreciation for the functions and limitations of BLAST are vital to using this program effectively. This unit will introduce the basic concepts behind BLAST, walk through BLAST searching protocols, and interpret common results. Curr. Protoc. Essential Lab. Tech. 1:11.1.1-11.1.36. © 2009 by John Wiley & Sons, Inc.

In the post-genomic era, more and more research projects involve the generation of molecular sequence data. How should these newly obtained DNA/protein sequences be analyzed, and how should they be prepared for submission to sequence databases? In this unit, we provide guidelines and a flow chart to help first-time users process new sequence data using third-party freeware programs and give a step-by-step demonstration on the preparation of a sequence file for submission to GenBank using the Sequin program. Curr. Protoc. Essential Lab. Tech. 1:11.2.1-11.2.20. © 2009 by John Wiley & Sons, Inc.

Risk assessment is a term that is applied to a process that we all use every day to make decisions. Conducting risk assessments while working in the laboratory should be integrated into the research culture. Reagents and equipment used in research laboratories can be hazardous if used improperly or without appropriate precautions. The first step in limiting risk is identifying the materials that can be hazardous. The second step is to assess the risk that they present during the experiments that will take place—a process called risk assessment. Once the hazards are identified and the risks assessed, a risk management strategy can be put in place to reduce the risks to an acceptable level. This process (hazard identification, risk assessment, risk management) must be repeated as often as new materials, new procedures, and new people are introduced into the laboratory. Curr. Protoc. Essential Lab. Tech. 1:A.1B.1-A.1B.11. © 2009 by John Wiley & Sons, Inc.

Phylogenetic trees represent hypotheses about evolutionary relationships between organisms or nucleotide or amino acid sequences. Because the best BLAST hit often does not represent the most closely related sequence, phylogenetic analyses are an essential extension of inquiry into any new protein or gene. In this unit, the reader will first learn how to create a multiple sequence alignment using ClustalX. He or she will then learn how to use that alignment to build a neighbor-joining phylogeny using the program Geneious. Finally, the user will learn how to interpret the phylogeny in light of the research questions. Curr. Protoc. Essential Lab. Tech. 1:11.3.1-11.3.17. © 2009 by John Wiley & Sons, Inc.

Spectroscopy is the study of the interaction of light with matter. By observing how light interacts with matter—through reflection, refraction, elastic scattering, absorbance, inelastic scattering, and emission—it is possible to (1) identify the wavelengths of light that interact with atoms and molecules, or (2) quantify the amount of light being absorbed, reflected, scattered, or emitted at a particular wavelength. This unit describes the background and basic principles of spectrophotometry (the study of the reflection or transmission properties of a substance as a function of wavelength, in particular, absorbance spectrophotometry. Also included is discussion of key spectrophotometer components and their functions, the relationship between absorbance and transmittance, experiment considerations, and the steps necessary in preparing a standard curve for determining absorbance concentration.

Bacteriophages (phages) are viruses that infect bacteria. Phages are the most abundant biological entities on Earth, and have influential effects on every ecosystem. Phage genomes represent a vast gene pool from which bacteria can draw upon for rapid evolution. Since the co-discovery of phages in 1915 by Frederick Twort and 1917 by Felix d'Herelle, their potential as subjects of laboratory research has been exploited to great effect. Phage research has resulted in important strides in biology, from the elucidation of the function of DNA and the discovery of messenger RNA to the study of basic molecular interactions and genetic regulation. These fascinatingly simple and diverse entities are also valuable as diagnostic tools, genetic screening vectors, and potential therapeutics. This article provides an overview of techniques essential for entering the world of bacteriophage study, and contains protocols for the propagation, maintenance, storage, and basic characterization of phages. Curr. Protoc. Essential Lab. Tech. 7:4.4.1-4.4.33. © 2013 by John Wiley & Sons, Inc.

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), investigate subunit compositions, verify homogeneity of protein samples, and purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). Support protocols cover the casting of gels, calculation of molecular mass using the electrophoretic mobility of a protein, and purification of SDS by recrystallization.