Current Protocols Essential Laboratory Techniques最新文献

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. © 2017 by John Wiley & Sons, Inc.

This appendix provides general techniques, equipment, and media used for the growth of many commonly encountered bacteria. For specific growth conditions, readers should refer to units regarding the laboratory maintenance of the organism of interest. © 2017 by John Wiley & Sons, Inc.

Fluorescent proteins have revolutionized biology by enabling what was formerly invisible to be seen clearly, and potentially enable imaging of all cells in living animals. There are numerous applications for in vivo imaging with fluorescent proteins, including use in cancer, immunology, infection, and neurobiology studies. This unit describes how introducing the GFP and RFP gene into malignant tissue has enabled imaging of primary cancer and the metastatic process with subcellular resolution in vivo. © 2017 by John Wiley & Sons, Inc.

It is well known that transcript localization controls important biological processes, including cell fate determination, cell polarity, cell migration, morphogenesis, neuronal function, and embryonic axis specification. Thus, the sub-cellular visualization of transcripts in ‘their original place’ (in situ) is an important tool to infer and understand their trafficking, stability, translation, and biological functions. This has been made possible through the use of labeled ‘anti-sense’ probes that can be readily detected after hybridization to their ‘sense’ counterparts. The following is a series of protocols for conducting in situ hybridization in Drosophila (fruit fly) embryos or tissues. Probe-detection methods include a relatively simple alkaline phosphatase reaction, as well as higher-resolution and higher-throughput versions using fluorescence-conjugated tyramide labeling. New modifications that enhance probe penetration and detection in various tissues are also provided. © 2017 by John Wiley & Sons, Inc.

In this appendix, we provide an outline of methods used in analyzing biological data. We give a summary of types of data encountered and the appropriate methods to apply for the questions of interest. Statistical techniques described include the t test, the Wilcoxon rank sum test, the Mann-Whitney-Wilcoxon test, ANOVA, regression, and the chi-square test. For each method, we give the appropriate assumptions, the details of the test, and a complete concrete example to follow. We also discuss related ideas such as multiple comparisons, why correlation does not imply causation, and ideas of sampling from a population. © 2017 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 and then will learn how to use that alignment to build a neighbor-joining phylogeny using the software program MEGA. Finally, the user will learn how to interpret a phylogeny in light of the research questions. © 2017 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 is vital to using this program effectively. This unit will introduce the basic concepts behind BLAST, walk through BLAST searching protocols, and interpret common results. © 2017 by John Wiley & Sons, Inc.

Blotting techniques are among the most common approaches used in a molecular biology laboratory. These techniques, Southern, northern, and immunoblotting, are applicable to a variety of macromolecules including DNA, RNA, and protein, respectively. Each of the techniques are dependent on the ability to resolve the individual macromolecules in a size-dependant manner, transfer the molecules to a solid support, and finally use a defined probe to detect the specific molecule of interest. The utilization of the blotting technology over the last 30 years has been instrumental to the elucidation of many fundamental biological processes. The continued use of blotting technology holds promise for even greater discovery over the next 30 years. © 2016 by John Wiley & Sons, Inc.

Chromatography is a widely used technique of separation which depends on the differential interaction of molecules between a stationary phase and a mobile phase. In most modern life science laboratories, purification of proteins plays an important role. Almost all types of laboratory-based liquid chromatography techniques used for purification of proteins are carried out with the stationary phase contained in tubular columns. Although individual methods for sample loading or elution of these columns are usually different depending on the specific technique used, this general experimental procedure that uses tubular columns is known as column chromatography. This unit is focused on setting up laboratory-based column chromatography methods for separation of proteins using three commonly used chromatographic techniques for separation of proteins, such as size exclusion, ion exchange, and affinity chromatography. © 2016 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. © 2016 by John Wiley & Sons, Inc.