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The Isolation of cDNAs by Hybridization of YACs to cDNA Libraries

The isolation of genes from large candidate regions is one of the major problems for the molecular biologist. With the advent of yeast artificial chromosomes (YACs), the problem of cloning these regions is now largely solved; however, screening these large genomic regions for expressed seq ...

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Transfection of Mammalian Cells via Lipofection

The use of baker’s yeast, Saccharomyces cerevisiae, for the cloning of extremely large genomic intervals (exceeding 1 Mb) was made possible with the development of yeast artificial chromosomes (YACs) (1). YACs are linear molecules containing all the control elements necessary for stab ...

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cDNA Selection with YACs

One of the major efforts in the field of positional cloning and the human genome project is to identify coding sequences or transcription units in large genomic regions such as yeast artificial chromosomes (YACs). The task proves to be challenging because only a small percent of the total DNA of the ge ...

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Markers, Selection, and Media in YAC Cloning

The genetic markers used in yeast, and in yeast artificial chromosome (YAC) cloning, are largely defined by the manipulation of growth media. This chapter, apart from providing recipes for media formulation, also contains a brief introduction to genetic markers in yeast that are relevant to Y ...

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YAC Library Screening I: Preparation of Hybridization Filters and PCR Pools

The storage of yeast artificial chromosome (YAC) libraries in ordered microtiter plates required a new approach to screening for clones containing specific DNA sequences. Screening libraries of some 60,000 clones by hybridization to filters prepared from individual 96-well micr ...

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YAC Library Screening II: Hybridization and PCR-Based Screening Protocols

Yeast artificial chromosome (YAC) libraries stored in microtiter plates are available for screening as either complex PCR pools or hybridization filters generated from YACs gridded at high densities (see Chapter 3). Different libraries may be available as either PCR pools, hybridiz ...

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Cloning of Human Telomeres in Saccharomyces cerevisiae

Telomeres are specialized structures that form the ends of eukaryotic chromosomes and that are required to fulfill a number of varied functions, see Biessman and Mason for recent review (1). First, they must protect the chromosome ends from degradation and from fusion and recombination with ...

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Purification of YAC-Containing Total Yeast DNA

There are many methods describing the preparation of DNA from Saccharomyces cerevisiae. The method described in this chapter is a modification of those described by Olson et al. (1) and Philippsen et al. (2). Although simpler techniques are available (3), this method routinely provides relat ...

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Restriction Analysis of YACs

Restriction enzyme analysis of yeast artificial chromosome (YAC)-cloned DNA allows direct comparison to genomic DNA, particularly in regions were pulsed-field gel electrophoresis (PFGE) maps are available, or to DNA cloned in other vectors (1,2). Furthermore, it allows the compari ...

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RARE-Cleavage Analysis of YACs

The ordering of yeast artificial chromosome (YAC) clones by sequence-tagged site (STS)-content mapping has proven an effective means of constructing large, contiguously cloned arrays of DNA, some of which span almost entire human chromosomes (1). This method requires that each YAC clone ...

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YAC Localization by Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) is a rapid procedure for mapping YACs on metaphase chromosomes and for identifying chimeric YACs that contain cocloned DNA fragments from different genomic regions.

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Determination of Foreign Gene Copy Number in Stably Transfected Cell Lines by Southern Transfer Analysis

The quantitative appraisal of the number of foreign gene copies integrated within the genomes of stably transfected cells is most conveniently performed using the strategy known as Southern blotting (1,2). First introduced by E. M. Southern in 1975 (2) the basic protocol involves the follow ...

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Evaluation of Extrachromosomal Gene Copy Number of Transiently Transfected Cell Lines

The short-term expression of DNA introduced into eukaryotic cells is now widely used to investigate the biological activities of cellular and viral genes or their products. A number of different transfection methods are in common use and can be broadly divided into two categories, based on the ...

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Use of Dpn I Restriction Enzyme to Assess Newly Replicated Gene Copies in Amplifiable Vector Systems

Bacterially propagated plasmid DNA can be transfected into established eukaryotic cell lines or primary cell cultures by a variety of techniques, such as electroporation (see Chapter 5, this vol) (1), scrape-loading (2), and DEAE dextran (see Chapter 3) or calcium phosphate mediated gene t ...

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Calcium Phosphate Mediated Gene Transfer into Established Cell Lines

DNA transfection is one of the most important techniques in molecular genetics. It is this technique that has made possible the dissection of complex eukaryotic genes and the characterization of the function of their components (1–7) as well as the isolation of particular genes on the basis of th ...

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Use of Polymerase Chain Reaction (PCR) to Detect Homologous Recombination in Transfected Cell Lines

When DNA is introduced into eukaryotic cells, it can be integrated into the genome by homologous or illegitimate recombination 1,2. Despite great efforts to gain insight into the molecular mechanisms, our understanding of the recombination process is still in its infancy. In the absence of a m ...

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Induction of Erythroid-Specific Expression in Murine Erythroleukemia (MEL) Cell Lines

Murine erythroleukemia (MEL) cell lines are erythroid progenitor cells derived from the spleens of susceptible mice infected with the Friend virus complex 1. These virally transformed cells are arrested at the proerythroblast stage of development and can be maintained in tissue cult ...

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Transfection of the ChloramphenicolAcetyltransferase Gene into Eukaryotic Cells Using Diethyl-Aminoethyl (DEAE)-Dextran

The study of gene regulation in eukaryotic cells involves a practical requirement for two distinct techniques: first, a transfection system, or more simply, a way of getting DNA into a cell; and second, a reporter system, which, as the name suggests, is a means of finding out what the transfected DNA does f ...

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Polybrene/DMSOAssisted Gene Transfer

Efforts to expand the current repertory of cell types amenable to transfection have often been thwarted by a common obstacle-namely, the low tolerance displayed by the recipient cells toward the gene-transfer regimen itself. As a result, several laboratories have turned to the use of synth ...

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Electroporation Technique of DNA Transfection

Electroporation is a simple and rapid procedure by which DNA may be transferred into cells. Essentially, a high voltage pulse is applied to a suspension of cells and DNA placed between electrodes in a suitable cuvet. It is thought that this pulse induces local areas of cell-membrane breakdown, or po ...

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