Current Protocols Essential Laboratory Techniques最新文献

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.

The ubiquitous use of digital imaging has greatly simplified and accelerated life science research. However, the ease of manipulating digital images also presents serious ethical issues. This unit presents guidelines and cautions describing best practices for use, analysis, and presentation of digital image data.

A poster is one method of informing the world about results from a research project. Whether serving as an aid for a presentation or standing alone, it should provide information about a project. Both substantive and stylistic qualities should contribute to effectively communicating one's conclusions. This appendix gives general guidelines and suggestions for poster content, layout, and design to assist in preparing an effective poster.

Quantification of immunohistochemistry (IHC) and immunofluorescence (IF) using image intensity depends on a number of variables. These variables add a subjective complexity in keeping a standard within and between laboratories. Fast Fourier Transformation (FFT) algorithms, however, allow for a rapid and objective quantification (via statistical analysis) using cell morphologies when the microscopic structures are oriented or aligned. Quantification of alignment is given in terms of a ratio of FFT intensity to the intensity of an orthogonal angle, giving a numerical value of the alignment of the microscopic structures. This allows for a more objective analysis than alternative approaches, which rely upon relative intensities. Curr. Protoc. Essential Lab. Tech. 7:9.5.1-9.5.12. © 2013 by John Wiley & Sons, Inc.

The measurement of pH is one of the most basic and necessary skills in a life science laboratory. The function and physical characteristics of biological molecules are highly sensitive to the pH of the environment. Common biological buffers must be prepared with the appropriate pH, usually close to neutral for most biological applications. This unit includes a discussion of the different pH instrumentation, notably pH electrodes, and their applications. It also relates basic pH measurement theory to critical parameters for the technique, and covers the correct use, handling, and storage of pH instruments.

Immunoblotting (also referred to as Western blotting) uses antibodies to probe for a specific protein in a sample bound to a membrane. Typically, a protein sample is first size separated via electrophoresis (e.g., SDS PAGE). However, antibodies used for specific protein detection are restricted by the polyacrylamide gel and, to make the separated proteins accessible, the proteins need to be moved out of the gel and bound to a rectangular sheet of PVDF or nitrocellulose membrane. In this second step, the membrane, cut to the same dimensions of the SDS gel (e.g., 10 x 10 cm), is then laid on the gel surface. The gel and membrane sandwich is then positioned in specialized blotting equipment that electrophoretically transfers the negatively charged proteins from the gel onto the membrane. The nitrocellulose or PVDF membrane binds the proteins as they move out of the gel, producing an exact replica, on the membrane surface, of the original protein gel separation. The proteins bind with high capacity and, in contrast to the polyacrylamide gel, are freely accessible to antibody reagents. The membrane is then blocked to prevent any nonspecific protein binding and visualized by specific antibodies to detect the presence or absence of a particular protein. For routine quantitation of a protein, the SDS PAGE separation is not always needed, and whole cell lysates or other complex mixtures are bound directly to the membrane for analysis using slot or dot blotting. Applications of immunoblotting are many, and include antibody characterization, diagnostics, gene expression and post translational modification analysis.

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 of accurate concentrations and to transfer quantitatively samples of liquid from one container to others, an array of glassware and plasticware is available for these operations ranging from a microliter to a liter in volume. Considerations of temperature, solvent compatibility, and safety also need to be taken into account.

The purification and concentration of nucleic acids have become routine procedures in most biology and molecular biology laboratories. This unit covers the basic principles and procedures for the isolation, purification, and manipulation of solutions of DNA or RNA. The basic DNA protocol, using phenol extraction and ethanol precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations ≤1 mg/ml. Purification of DNA using commercially available silica membrane spin columns is presented as an alternate protocol. Isolation and purification of RNA from mammalian cells or tissues is also examined. Use of the protein denaturant guanidine thiocyanate and water-saturated phenol, followed by concentration by isopropanol precipitation, for producing small samples of RNA is illustrated in the basic RNA protocol.

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.

Real-time PCR is a recent modification to the polymerase chain reaction that allows precise quantification of specific nucleic acids in a complex mixture by fluorescent detection of labeled PCR products. Detection can be accomplished using specific, as well as nonspecific fluorescent probes. Real-time PCR is often used in the quantification of gene expression levels. Prior to using real-time PCR to quantify a target message, care must be taken to optimize the RNA isolation, primer design, and PCR reaction conditions so that accurate and reliable measurements can be made. This short overview of real-time PCR discusses basic principles behind real-time PCR, some optimization and experimental design considerations, and how to quantify the data generated using both relative and absolute quantification approaches. Useful Web sites and texts that expand upon topics discussed are also listed.