bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Newly Transcribed RNA
Authors: Greenberg Michael E. , Bender Timothy P. 
Published: June 2004

AbstractNewly transcribed RNA can be identified using the nuclear runoff transcription assay. Isolated nuclei, free of membranes and cytoplasmic debris, are required for the assay. Cell lysis that does not allow the isolation of nuclei free of cell membranes and cytoplasmic material often results in poor incorporation of 32P‐labeled UTP into nascent transcripts. Although there is no way to predict what cell types present this problem, many adherent cell lines and lymphocytes isolated from murine spleen or thymus do. Interestingly, very few nonadherent cell lines have posed this problem. As described in this unit, isolating nuclei by detergent lysis of cells works well for many tissue culture cell lines but may not be appropriate for all cell lines and many tissues. Protocols for detergent lysis and Dounce homogenization or cell lysis in an isoosmotic solution and centrifugation through a sucrose cushion are described along with a for the preparation of the cDNA nitrocellulose filter strips that are used to detect the presence of specific transcripts in the nuclear runoff transcription assay.
Size Fractionation Using Sucrose Gradients
Authors: Weis John H. , Quertermous Thomas 
Published: June 2004

AbstractDNA fragments migrate through a linear sucrose gradient at a rate that is dependent on their size. This procedure described in this unit provides good resolution for DNA fragments 5 to 60 kb in size, and sucrose gradients are also useful for purification of bacteriophage l vector arms. Partially digested genomic DNA can be fractionated for the production of cosmid or bacteriophage libraries and completely digested DNA can be fractionated for subgenomic DNA libraries. Protocols are provided in this unit for both partial and complete enzyme digestion of genomic DNA.
Production of a Complete cDNA Library
Authors: Klickstein Lloyd B. 
Published: June 2004

AbstractA complete cDNA library is one that contains at least one cDNA clone representing each mRNA in the cell. A method is provided in this unit for calculating the required base of a cDNA library such that the library should contain at least one copy of every mRNA. Either a phage or plasmid vector can be used in constructing a complete cDNA library, and protocols are described for both. Procedures for evaluating these cDNA libraries are also provided along with a procedure for evaluating the quality of the λ arms used in constructing complete phage cDNA libraries.
Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
Authors: Osoegawa Kazutoyo , de Jong Pieter J. , Frengen Eirik , Ioannou Panayiotis A. 
Published: June 2004

AbstractLarge‐insert genomic libraries are necessary for physical mapping of large chromosomal regions, for isolation of complete genes, and for use as intermediates in DNA sequencing of entire genomes. Construction of BAC and PAC libraries is detailed in the unit, including preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, dephosphorylation with alkaline phosphatase, and purification through pulsed‐field gel electrophoresis (PFGE). For the preparation of high‐molecular weight DNA for cloning, procedures for embedding total genomic DNA from lymphocytes or animal tissue cells are also provided. Other protocols detail partial digestion of genomic DNA with MboI or with a combination of EcoRI endonuclease and EcoRI methylase (including methods for optimizing the extent of digestion), and subsequent size fractionation by preparative PFGE. Finally, the isolation of BAC and PAC plasmid DNA for analyzing clones is also presented.
Plating and Transferring Cosmid and Plasmid Libraries
Authors: Weis John H. 
Published: June 2004

AbstractThere are two commonly used protocols for the screening of recombinant bacteria with hybridization probes. One method involves the spreading of bacteria on the surface of agar using a sterile spreader. A nitrocellulose membrane filter is then placed on top of the colonies and most of each colony is transferred to the filter. This method works well when relatively small numbers of positive colonies are being selected (up to several thousand). A second method, described in this unit, employs a matrix of some type (here nitrocellulose filters are used) upon which bacteria can be plated and grown when the filter is placed on top of a nutrient agar surface. Once the plated bacteria have grown into visible colonies, the filters can be used for replica plating and in situ hybridization analysis.
Using DNA Fragments as Probes
Authors: Strauss William M. 
Published: June 2004

AbstractAll hybridization methods depend upon the ability of denatured DNA to reanneal when complementary strands are present in an environment near but below their Tm (melting temperature). A detailed procedure is described in which bacteriophage plaques or bacterial colonies bound to a filter membrane are detected by hybridization with a radioactive probe. Hybridization proceeds on prewet filters placed in a sealable plastic bag. After hybridization the filters are removed from the sealed bag, excess probe is washed off, and the filters are autoradiographed to identify the clones that have hybridized with the probe. An differs mainly in that formamide is not used in the hybridization solution.
Immunoscreening of Fusion Proteins Produced in Lambda Plaques
Authors: St. John Thomas P. 
Published: June 2004

AbstractScreening large numbers of plaques containing particular proteins is accomplished by techniques that are analogous to those described for screening with radioactive DNA probes. However, in the basic protocol described here, the plaques are screened with antibodies specific to the desired proteins. An alternate protocol provides a method for increasing the amount of recombinant protein in each plaque by inducing expression from the lac promoter that directs its expression.
DNA Sequencing by the Dideoxy Method
Authors: Slatko Barton E. , Albright Lisa M. , Tabor Stanley , Ju Jingyue 
Published: June 2004

AbstractIn the basic dideoxy sequencing reaction, an oligonucleotide primer is annealed to a single‐stranded DNA template and extended by DNA polymerase in the presence of four deoxyribonucleoside triphosphates (dNTPs), one of which is 35S‐labeled. The reaction also contains one of four dideoxyribonucleoside triphosphates (ddNTPs), which terminate elongation when incorporated into the growing DNA chain. After completion of the sequencing reactions, the products are subjected to electrophoresis on a high‐resolution denaturing polyacrylamide gel and then autoradiographed to visualize the DNA sequence. Three variations of the dideoxy sequencing procedure are currently in use and are presented in this unit. In the “labeling/termination“ procedure, primer chains are initially extended and labeled in the absence of terminating ddNTPs, whereas in the traditional “Sanger” procedure, labeling and termination of primer chains occur in a single step. A recent variation of the dideoxy sequencing method is thermal cycle sequencing in which the reaction mixture, containing template DNA, primer, thermostable DNA polymerase, dNTPs, and ddNTPs, is subjected to repeated rounds of denaturation, annealing, and elongation steps. The resulting linear amplification of the sequencing products allows much less template DNA to be used and eliminates independent primer annealing and template denaturation steps, which are required for the labeling/termination or Sanger procedures. The use of automated fluorescent sequencers ofr four‐color dideoxy DNA sequencing is also described in detail.
Mutagenesis with Degenerate Oligonucleotides: Creating Numerous Mutations in a Small DNA Sequence
Authors: Hill David E. 
Published: June 2004

AbstractThe procedure described in this unit is an efficient method for using the products of a single oligonucleotide synthesis to create numerous mutations in a small region of DNA (20 to 80 nucleotides). Mutant oligonucleotides are generated during the organic synthesis reaction by including low concentrations of the three non‐wild‐type nucleotide precursors with the wild‐type precursor at each step of the synthesis. The product of such a DNA synthesis is a degenerate oligonucleotide, i.e., a complex mixture of related molecules, each of which has a defined probability of being altered from the wild‐type sequence. The frequencies and types of zero, single, double, and higher order mutations can be set simply by choosing the appropriate amounts of non‐wild‐type precursors at each step of nucleotide addition during the synthesis.
Random Mutagenesis by PCR
Authors: Wilson David S. , Keefe Anthony D. 
Published: June 2004

AbstractError‐prone PCR (EP‐PCR) is the method of choice for introducing random mutations into a defined segment of DNA that is too long to be chemically synthesized as a degenerate sequence. Using EP‐PCR, the 5′ and 3′ boundaries of the mutated region may be defined by the choice of PCR primers. Accordingly, it is possible to mutagenize an entire gene or merely a segment of a gene. The average number of mutations per DNA fragment can be controlled as a function of the number of EP‐PCR doublings performed. The EP‐PCR technique described here is for a 400‐bp sequence, and an is for a library. EP‐PCR takes advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the addition of Mn2+, increasing the Mg2+ concentration, and using unequal dNTP concentrations.
Directed Mutagenesis Using the Polymerase Chain Reaction
Authors: Cormack Brendan 
Published: June 2004

AbstractThis unit contains two basic protocols for introducing base changes into specific DNA sequences. The first describes the incorporation of a restriction site and the second details the generation of specific point mutations. An describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.
Direct Analysis of RNA after Transfection
Authors: Kingston Robert E. 
Published: June 2004

AbstractIt is possible to transfect mammalian cells with a gene of interest and directly detect the RNA made from that gene 24 to 48 hr later. The ability to analyze the RNA directly allows mutation and functional analysis of an intact gene, as the promoter does not have to be subcloned. This technique is also essential when fusion genes are used. In order to be sure that the level of the reporter protein produced by a fusion gene provides an accurate measure of appropriately initiated RNA from the promoter under study, it is necessary to determine the amount and 5′ end of the fusion message. An investigator who verifies this using direct RNA analysis can proceed with some confidence that the level of the reporter protein is a measure of promoter activity. The difficulty in directly analyzing RNA is that the sensitivity of detection is at the limits of present technology. Consequently, the transfection protocol, RNA preparation, and RNA analysis techniques all have to be working near optimum in order for an experiment to work. This overview discusses parameters that must be considered when directly analyzing RNA after a transfection: transfection efficiency, the method of preparation of the RNA, the method of analysis of RNA, and strength of the promoter.
Overview of the Retrovirus Transduction System
Authors: Cepko Constance , Pear Warren 
Published: June 2004

AbstractA retrovirus vector is an infectious virus used to introduce a nonviral gene into mitotic cells in vivo or in vitro. The efficient and precise integration machinery of naturally occurring retroviruses is utilized to produce either a single copy or a few copies of the viral genome stably integrated into a host chromosome. This unit presents an overview of the retrovirus life cycle and a description of vector designs and packaging cell lines.
Preparation of a Specific Retrovirus Producer Cell Line
Authors: Cepko Constance 
Published: June 2004

AbstractEstablishing a cell line that produces high levels of a specific retrovirus construct involves several steps. First, the retroviral construct must be stably introduced into an appropriate packaging cell line. This can be accomplished either directly by transfection or by transfection to produce a transient virus stock followed by cross‐infection of this stock into a separate packaging line; procedures for both approaches are described in this unit. After stable lines are produced, they must be characterized to identify lines that produce high titers of virus with an appropriate structure. A drug selection protocol (the most common method for determining virus titer) is provided along with a sample calculation of BAG virus titer. Sometimes, a short and direct method of estimating virus titer is available when the virus encodes a histochemically detectable gene such as lacZ. For this case, an Xgal staining protocol is described. Similarly, a protocol is provided to estimate the levels of virus being produced by a given cell line by quantitating any specific product of the virus (e.g., RNA) present within the producer line.
Transient Transfection Methods for Preparation of High‐Titer Retroviral Supernatants
Authors: Pear Warren 
Published: June 2004

AbstractGeneration of high‐titer retrovirus by transient production not only is less laborious than production of stable retroviral producer cell lines, but also has allowed the production of high‐titer retroviral supernatants from cDNAs that cannot be achieved by stable producer cell lines. Transient transfection has also increased the versatility of retrovirus‐mediated gene transfer to include the rapid testing of different constructs, viral pseudotyping, and construction of retroviral cDNA libraries. Systems based on human 293 cells, an adenovirus‐transformed human embryonic kidney cell line have produced the highest retroviral titers and are the most widely used. This unit describes methods for optimizing retroviral production from the 293‐based systems and for growing and freezing 293 cells. Methods are included for pseudotyping the virus with VSV G protein by sequential transfection or cotransfection. Virus produced by transiently transfected cells can be used to infect cells. Protocols are provided for infection of adherent cells either directly with retroviral supernatant or by spin infection. In addition, procedures are included for infection of nonadherent cells by addition of retrovirus supernatant, cocultivation with producer cells, or spin infection. These infection methods are also applicable to retrovirus produced by any of the stable producer cell lines.
Large‐Scale Preparation and Concentration of Retrovirus Stocks
Authors: Cepko Constance 
Published: June 2004

AbstractFor some applications, such as infection of cells in vivo, it is necessary to concentrate retrovirus stocks in order to increase their titer. Because viruses are macromolecular structures, they can be concentrated fairly easily by a relatively short centrifugation step. This unit provides protocols in which the viral particles are either pelleted or centrifuged onto a sucrose density step gradient. An alternate protocol details how virions can be precipitated using polyethylene glycol or ammonium sulfate, and the resulting precipitate collected by centrifugation. After resuspension of the precipitates, the virions can be used directly, or further purified either by sedimentation onto sucrose density gradients, or by column filtration as described here. Perhaps most simply of all, an alternate procedure describes how small volumes of virus stock can be concentrated by centrifugation through a filter that allows only small molecules to pass.
Retrovirus Infection of Cells In Vitro and In Vivo
Authors: Cepko Constance , Pear Warren 
Published: June 2004

AbstractThere are many applications in which retrovirus vectors are used as transduction agents. In some cases, the vector carries a gene that one wishes to express in a target cell in order to study the function of that gene. In other cases, the virus is used to introduce a histochemical marker gene into cells in order to follow their fate. Retrovirus vectors can also be used in a variety of cells type to investigate regulatory sequences in which a reporter gene and regulatory sequences are carried by the vector and to immortalize or transform primary cells by transduction of oncogenes. For each application, the infection protocol may vary and must often be optimized. Guidelines for infection of cells in some typical in vivo and in vitro experiments are presented in this overview.
Human Somatic Cell Gene Targeting
Authors: Waldman Todd , Lee Carolyn , Nishanian Tagvor G. , Kim Jung‐Sik 
Published: June 2004

AbstractHuman somatic cell gene targeting provides a powerful tool to scientists studying gene function in cultured human cells. This technology allows scientists to knock out genes in human somatic cells in a fashion analogous to the creation of knockout mice. Human somatic cell gene targeting brings the power of genetics to the study of human genes in human cells by making it possible to compare cells or individuals that are genetically identical except for a single, well‐defined mutation in an endogenous gene. These modified cells can be studied both in vitro and in vivo. This unit presents protocols for human somatic cell gene targeting.
One‐Dimensional SDS Gel Electrophoresis of Proteins
Authors: Gallagher Sean R. 
Published: June 2004

AbstractElectrophoresis is used to separate complex mixtures of proteins, (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates) to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in the gel matrix; pore size decreases with higher acrylamide concentrations. The combination of gel 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, that is, in the presence of sodium dodecyl sulfate (SDS). Both full‐size and minigel formats are detailed. Several alternate protocols are provided for specific applications. The first two alternate protocols cover electrophoresis of peptides and small proteins, separations that require modification of standard buffers: either a Tris‐tricine buffer system, or modified Tris buffer in the absence of urea. Continuous SDS‐PAGE is a simplified method in which the same buffer is used for both gel and electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and electrophoresis of various types of gels: ultrathin gels, multiple single‐concentration gels, gradient gels, multiple gradient gels, and multiple gradient minigels.
Overview of Digital Electrophoresis Analysis
Authors: Medberry Scott , Gallagher Sean , Moomaw Butch 
Published: June 2004

AbstractGel electrophoresis has become a ubiquitous method in molecular biology for separating biomolecules. This prominence is the result of several factors, including the robustness, speed, and potentially high throughput of the technique. The results of this method are traditionally documented using silver halide‐based photography followed by manual interpretation. While this remains an excellent method for qualitative documentation of single‐gel results, digital capture offers a number of significant advantages when documentation requires quantitation and sophisticated analysis. Digital images of gel electropherograms can be obtained rapidly using an image‐capture device, and the images can be easily manipulated using image analysis software. This overview presents reasons for digital documentation and analysis, defines some important key terms for imaging, explains the capture process and reviews the devices used for image capture, and provides an introduction to the software and methods used for one‐ and two‐dimensional digital image analysis.
Detection of Proteins on Blot Transfer Membranes
Authors: Sasse Joachim , Gallagher Sean R. 
Published: June 2004

AbstractIn the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.
HPLC of Peptides and Proteins: Preparation and System Set‐Up
Authors: Boysen Reinhard I. , Hearn Milton T.W. 
Published: June 2004

AbstractThe eight basic modes of HPLC currently in use for peptide and protein analysis and purification are presented in this overview. They include: size‐exclusion chromatography (HP‐SEC), ion‐exchange chromatography (HP‐IEX), normal phase chromatography (HP‐NPC), hydrophobic interaction chromatography (HP‐HIC), reversed‐phase chromatography (RP‐HPLC), hydrophilic interaction chromatography (HP‐HILIC), immobilized metal ion affinity chromatography (HP‐IMAC), and biospecific/biomimetic affinity chromatography (HP‐BAC). In addition, some subsets of these chromatographic modes, e.g., mixed mode chromatography (HP‐MMC), charge transfer chromatography (HP‐CTC), or ligand‐exchange chromatography (HP‐LEC) are described. The unit includes overviews of the system requirements and planning strategies for set‐up of the systems.
Metabolic Labeling with Amino Acids
Authors: Bonifacino Juan S. 
Published: June 2004

AbstractMetabolic labeling techniques are used to study biosynthesis, processing, intracellular transport, secretion, degradation, and physical‐chemical properties of proteins. This update focuses on pulse‐labeling and pulse‐chase experiments done with
[35S]‐methionine, but gives directions for labeling with other radiolabeled amino acids, and also offers guidance for the safe use and handling of 35S‐labeled compounds.
Isolation of Proteins for Microsequence Analysis
Authors: Moss Malcolm 
Published: June 2004

AbstractThis unit presents a method for determining the amino‐terminal sequence of polypeptides or proteins. It is particularly appropriate for large fragments of insoluble or hydrophobic proteins or proteins that cannot be purified to >90% molar purity without electrophoresis. If the protein is blocked at the amino terminus, chemical cleavage or partial enzymatic digestion must be performed prior to electrophoresis. Upon isolation, the internal amino acid sequence is analyzed as described.
Overview of Peptide and Protein Analysis by Mass Spectrometry
Authors: Carr Steven A. , Annan Roland S. 
Published: June 2004

AbstractThe goals of this unit are to familiarize peptide and protein chemists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide. The emphasis here is on established tools and techniques that can realistically be used for problem solving. As a result, many useful instruments and techniques employed by the trained mass spectroscopist are not discussed here, because they do not satisfy these criteria or are not yet widely available. A few of these are briefly mentioned at the end of this introductory overview. The overview concludes with a tutorial discussion on the meaning and practical importance of a number of fundamental experimental and performance‐related issues that have important implications for interpretation and use of mass spectral data, such as mass accuracy and resolution.