Current Protocols Essential Laboratory Techniques最新文献

Thin layer chromatography (TLC) is a quick, sensitive, and inexpensive technique used to determine the number of components in a mixture, verify the identity and purity of a compound, monitor the progress of a reaction, determine the solvent composition for preparative separations, and analyze the fractions obtained from column chromatography. This unit is mainly aimed at novice experimenters, describing in detail the strategies and principal steps for performing a TLC experiment, with illustrations of the relevant instruments, as well as approaches for obtaining and understanding results. Valuable practical tips and troubleshooting solutions are also provided throughout the unit. Curr. Protoc. Essential Lab. Tech. 8:6.3.1-6.3.18. © 2014 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. 8:11.1.1-11.1.35. © 2014 by John Wiley & Sons, Inc.

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. Curr. Protoc. Essential Lab. Tech. 8:10.3.1-10.3.40. © 2014 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, experimental considerations, and the steps necessary in preparing a standard curve for determining absorbance concentration. Curr. Protoc. Essential Lab. Tech. 6:2.1.1-2.1.28. © 2012 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. Curr. Protoc. Essential Lab. Tech. 6:7.3.1-7.3.28. © 2012 by John Wiley & Sons, Inc.

E.M. Southern invented blotting of DNA in 1975; the method was extended to RNA and named northern blotting in 1977. Southern blotting includes limited depurination, denaturation, and neutralization of the DNA in gels (where they have been separated in size by electrophoresis) and capillary transfer of the DNA onto nitrocellulose or nylon blotting membranes. For northern blotting, RNA is guarded from basic pH and RNase, denatured, separated by electrophoresis, and then blotted on to nylon blotting membranes. Either type of blot is then blocked to prevent nonspecific binding, hybridized with probe, and washed. Next, the sequences of interest are located by detecting labeled probes. One alternative method involves dot/slot blotting when the size of the nucleic acid being probed is not of interest. Also, electrophoretic transfer from polyacrylamide gels can be used when the nucleic acid fragments of interest are too small to be resolved on agarose gels. Artifacts in Southern blot can result from incomplete digestion, overloading the blotting membrane, incomplete blocking, damaged blot media, and air bubbles. In northern blotting, RNA quality must be monitored, and RNA that is degraded or contaminated with excess DNA should be avoided. Curr. Protoc. Essential Lab. Tech. 6:8.2.1-8.2.26. © 2012 by John Wiley & Sons, Inc.

Chromophore-binding domains from plant and bacterial photoreceptors have received increasing interest as new sources of genetically encoded fluorescent proteins (FPs). In particular, FPs based on the flavin-binding LOV (Light, Oxygen, or Voltage sensing) domain offer advantages over green fluorescent protein (GFP) owing to their smaller size and utility under anaerobic conditions. Recombinant expression of LOV domains in Escherichia coli (E. coli) is fast, easy to detect, and inexpensive given the innate ability of LOV domains to acquire their ubiquitous organic cofactor from the cellular environment. This manuscript describes the strategies and variables to consider when expressing and purifying LOV-domain protein fusions from liquid cultures of E. coli. Strategies for expressing and visualizing LOV-domain fusion proteins in E. coli grown on agar medium are also described. Curr. Protoc. Essential Lab. Tech. 6:13.1.1-13.1.11. © 2012 by John Wiley & Sons, Inc.

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. Curr. Protoc. Essential Lab. Tech. 6:5.2.1-5.2.21. © 2012 by John Wiley & Sons, Inc.

This unit describes protocols for detecting proteins in SDS-polyacrylamide gels. It describes methods for Coomassie blue and silver staining, as well as the fluorescent stains SYPRO Orange and Red. Staining with Coomassie blue is easier and more rapid; however, silver staining methods are considerably more sensitive and can thus be used to detect smaller amounts of protein. Alternative rapid staining procedures are provided for each method. Fluorescent staining is a popular alternative to the traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Curr. Protoc. Essential Lab. Tech. 6:7.4.1-7.4.14. © 2012 by John Wiley & Sons, Inc.

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. Curr. Protoc. Essential Lab. Tech. 5:4.3.1-4.3.32. © 2011 by John Wiley & Sons, Inc.