Current Protocols Essential Laboratory Techniques最新文献

Mammalian cell culture is the process of growing animal cells in vitro in a flask or dish. This unit describes the methods, equipment, supplies, and reagents used in a cell culture laboratory. Because preventing contamination and maintaining purity of cell cultures is arguably the greatest challenge in animal cell culture, the principles of aseptic technique in mammalian cell culture are presented in detail. Cell culture media and additives are discussed. A method of counting cells and assessing their viability with a hemacytometer is illustrated. The steps needed to maintain an adherent cell line by feeding and passing cells from one culture vessel to another are presented, and variations for passing suspension-grown cells are also outlined. Long-term cryogenic storage of cells in liquid nitrogen is discussed and a protocol for freezing cells is provided, as are details for thawing and recovering viable cells from frozen stocks. © 2015 by John Wiley & Sons, Inc.

Agarose gel electrophoresis, which separates and sizes linear DNA and RNA fragments, is arguably the most basic and essential technique in molecular biology. It is commonly employed for analysis of PCR products, plasmid DNA, and products of restriction enzyme digestion. It is the first step for analysis of specific DNA and RNA fragments by northern and Southern blots. In this unit, we provide both written instructions and photographic images to take the reader from preparing a first agarose gel to analyzing results and determining the size of sample DNA. We include two protocols: agarose gel electrophoresis (commonly used to analyze DNA) and denaturing gel electrophoresis (for analyzing RNA). We have divided each protocol into four basic steps: (1) preparing and pouring the agarose gel; (2) preparing and loading samples; (3) running the agarose gel; and (4) staining the gel using the fluorescent stain ethidium bromide to visualize DNA and RNA. © 2015 by John Wiley & Sons, Inc.

The visualization of fluorescently tagged molecules is a powerful strategy that can contribute to the understanding of the complex dynamics of the cell. A particularly robust and broadly applicable method is immunofluorescence microscopy, in which a specific fluorescently labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, including the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine-tuning depending on the cell type, the antibody, and the antigen, there are steps common to nearly all applications. This unit provides protocols for visualization of the cytoskeleton in two very different kinds of cells: flat, adherent fibroblasts and thick, free-swimming Tetrahymena cells. © 2015 by John Wiley & Sons, Inc.

This unit describes the common types of volumetric apparatus used in the life science laboratory, their use, and care. When an experimenter needs to prepare solutions at accurate concentrations and quantitatively transfer samples of liquid from one container to another, an array of glassware and plasticware is available for these operations ranging from a microliter to more than a liter in volume. Considerations of temperature, solvent compatibility, and safety also need to be taken into account. © 2015 by John Wiley & Sons, Inc.

One of the most important requirements for success with any biochemical protocol is the accurate preparation of reagents. This unit provides general guidelines for the preparation of reagents, including use of clean glass- and plasticware, use of high-purity reagents, safe handling of chemicals and biochemicals, use of high-purity distilled or deionized water, and avoidance of chemical or microbial contamination. Different units of concentration used for preparation of reagents or buffer solutions are described. This unit also includes a section for the preparation of buffer solutions with a practical example. © 2014 by John Wiley & Sons, Inc.

Centrifugation is a ubiquitous technique in the life science laboratory that uses specialized equipment (centrifuges) to spin, or centrifuge, samples in rotors, generating centrifugal force for a range of applications that include pelleting of precipitates, isolating organelles, and protein and DNA purification. The unit describes basic applications, definitions, and references, and provides up-to-date tables for rotor specifications from major suppliers. © 2014 by John Wiley & Sons, Inc.

Digital imaging is the method of choice for documentation and analysis of electrophoretic separations of protein and DNA. Digital images of gel electropherograms can be obtained rapidly using CCD-based cameras, and the images can be easily archived and analyzed using image analysis software. This overview defines important key terms and calculations for imaging, explains the capture process, reviews the range of CCD technologies used for image capture, and provides an introduction to the software and methods used for one- and two-dimensional digital image analysis. © 2014 by John Wiley & Sons, Inc.

ImageJ is a freely available, cross-platform (e.g., Windows, Mac, Linux) image processing and analysis program developed by the NIH. In addition to being readily available for no cost, ImageJ is supported by a wide range of constantly evolving user-created functionalities to address a remarkable range of applications, complementing commercial software that typically comes with imaging instruments such as digital gel-imaging systems or microscopy workstations. New processing/analysis macros and plug-ins are routinely added to the support site, and are frequently validated via refereed publications. With the continued improvements and growth of fluorescence-based applications, ImageJ continues to be a mainstay in the laboratory. ImageJ has extensive support materials available online, its base code is regularly updated, and a survey of Medline references indicates that it is one of the most widely used image-analysis packages available today. © 2014 by John Wiley & Sons, Inc.

This manual is a collection of basic techniques central to the study of nucleic acids, proteins, and whole-cell/subcellular structures. The following is an overview of how the basic techniques described in this manual can be used alone or in sequence to answer questions about the properties of proteins and nucleic acids. Flowcharts are provided to orient the novice researcher in the use of fundamental molecular techniques, and provide perspective regarding applications of the technical units in this manual.

Listed below are common questions about nucleic acids and techniques used to answer them. Also refer to Figure 1.

Listed below are common questions about proteins and techniques used to answer them. Also refer to Figure 2.

For many experiments, the concentration of protein in the sample must first be quantified ( UNIT here). For example, this is often done prior to performing SDS-PAGE and/or an immunoblot to ensure equal loading of different protein samples for comparison. To determine the localization of specific proteins, cells can first be lysed and fractionated by centrifugation ( UNIT here), followed by immunoblotting of the proteins ( UNIT here) from fractions containing specific cell substructures. A chromatography step would further resolve proteins from the various fractions ( UNITS here & here). Alternatively, localization of specific proteins to distinct cellular structures can be done using the immunofluorescence technique ( UNIT here).

This manual also includes techniques for studying whole cells and their substructures. These include cell fractionation by centrifugation ( UNIT here), cell imaging by conventional light microscopy ( UNIT here), and imaging by fluorescence microscopy ( UNIT here). Refer to Figure 3. All of these protocols require first growing cells in culture. Protocols for culturing bacteria ( UNIT here), culturing mammalian cells ( UNIT here), and culturing yeast ( UNIT here) are provided.

These techniques can be used to answer questions such as:

Does cell morphology change under different treatment conditions?

Does cell behavior change under different treatment conditions?

Do genetically altered cell lines display morphological phenotypes?

In which cellular substructures d

This unit is an overview of the subsequent units on protein and nucleic acid electrophoresis separation techniques. Curr. Protoc. Essential Lab. Tech. 8:7.1.1-7.1.7. © 2014 by John Wiley & Sons, Inc.