Current Protocols Essential Laboratory Techniques最新文献

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in molecular biology. Two methods for direct absorbance measurements at 260 nm are described—the first is a traditional cuvette-based method, and the second is a microvolume method that requires no cuvettes or capillaries. In addition, three fluorescence techniques using Hoechst 33258, ethidium bromide, and PicoGreen reagent are presented in this unit. These five procedures cover a range from 5 to 10 ng/ml DNA to 15,000 µg/ml DNA. Reliable quantitation of proteins is possible using several types of assays. UV spectroscopy is the simplest approach but is limited in sensitivity. More sensitive assays that use Coomassie blue binding, bicinchoninic acid (BCA), and the Lowry reaction are also described. All assays are prone to amino acid composition errors and interference from assay solution components. Flow charts and tables to help with appropriate method selection are included. Curr. Protoc. Essential Lab. Tech. 5:2.2.1-2.2.36. © 2011 by John Wiley & Sons, Inc.

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. Specialized blotting equipment 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 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. Applications of immunoblotting are many, and include antibody characterization, diagnostics, gene expression, and post-translational modification analysis. Curr. Protoc. Essential Lab. Tech. 4:8.3.1-8.3.36. © 2010 by John Wiley & Sons, Inc.

The synthesis of specific recombinant proteins using single-celled organisms from bacteria to mammalian tissue culture cells has become a major source of biopharmaceutical products for the industry and a source of a wide variety of proteins for academic research. A range of organisms are utilized for this purpose. One of the newest and most promising of these is the yeast Pichia pastoris. This article provides detailed basic protocols for the expression of heterologous genes and the synthesis of recombinant proteins utilizing this yeast. Specifically provided are protocols for the insertion of foreign vector DNAs into the yeast by electroporation, amplification of vector sequences by the post-translational vector amplification (PTVA) method, and growth and expression of foreign genes in shake flask cultures. Curr. Protoc. Essential Lab. Tech. 4:13.2.1-13.2.14. © 2010 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 embryos or tissues. These methods include standard alkaline phosphatase methods, as well as higher resolution and throughput variations using fluorescence-based probe detection. New modifications that enhance probe penetration and detection in various tissues are also provided. Curr. Protoc. Essential Lab. Tech. 4:9.3.1-9.3.24. © 2010 by John Wiley & Sons, Inc.

The goal of this appendix is to introduce basic methods in graphing and data analysis and explore some fundamental concepts in statistical reasoning. Three examples of data-analysis problems relevant to molecular biology are used to illustrate methods covered in a first statistics course, including the two-sample t test, simple linear regression, and chi square tests for goodness of fit and contingency table hypotheses. The appendix also explores the selection and interpretation of appropriate summary graphs for these analyses, including the use of error bar plots, scatterplots, and bar charts. In addition, a number of key terms and concepts are introduced and explained in the context of the three example problems, including summary statistics, sampling variation, the standard error, null hypothesis testing, the use of test statistics, and the interpretation of p values. Curr. Protoc. Essential Lab. Tech. 3:A.4B.1-A.4B.22. © 2010 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. Curr. Protoc. Essential Lab. Tech. 3:A.3C.1-A.3C.24. © 2010 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. It also relates basic pH measurement theory to critical parameters for the technique, and covers the correct use, handling, and storage of pH instruments. Curr. Protoc. Essential Lab. Tech. 3:3.2.1-3.2.16. © 2010 by John Wiley & Sons, Inc.

Fluorescence is an extremely powerful tool in modern biology, physics, and chemistry laboratories. This unit focuses on methods that can be applied to biological studies. Starting with the preparation of fluorescently conjugated antibodies, the first part explains the physical principles of fluorescence and the features in an absorption spectrum, and how these are used to measure the degree of labeling on conjugated antibodies. The protocols describe how to obtain and interpret a fluorescence emission spectrum and utilize this information as a fluorescence indicator for protein detection. Curr. Protoc. Essential Lab. Tech. 2:2.4.1-2.4.29. © 2009 by John Wiley & Sons, Inc.

DNA sequencing, the process of determining the precise order of nucleotides in a DNA strand, is a fundamental and ubiquitous method in molecular biology. Examples of its many uses include characterization of unknown DNA, mutation detection, allele identification, and clone construct confirmation. Advances in sequencing technology, motivated by the successful International Human Genome Project, have resulted in unprecedented access to high-quality and cost-effective automated analysis. It is increasingly common for researchers to delegate routine DNA sequencing needs to dedicated laboratories or companies specializing in this technology. In this unit, we discuss key protocols for maximizing the success of DNA sequencing when working with such service providers and describe detailed troubleshooting advice for analyzing anomalous results. Curr. Protoc. Essential Lab. Tech. 2:12.1.1-12.1.19. © 2009 by John Wiley & Sons, Inc.