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Proteomics-Based Method for Risk Assessment of Peroxisome Proliferating Pollutants in the Marine Environment

Pollution in aquatic environment is of increasing concern for its impact on both human and natural populations. Applying proteomics to monitor marine pollution is a new approach to evaluate the effects of environmental pollutants on the biota. Aquatic organisms living in coastal and est ...

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The MIRA Method for DNA Methylation Analysis

DNA methylation patterns are often altered in human cancer and aberrant methylation is considered a hallmark of malignant transformation. Several methods have been developed for the characterization of gene-specific and genome-wide DNA methylation patterns. In this chapter, we ...

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Immunodetection Array

A novel procedure for DNA methylation analysis is described that characterizes the extent of DNA methylation in CpG islands. The basic concept relies on direct immunodetection of 5′ methylcytosines (5′ mCs) without the need for bisulfite treatment utilizing a microarray format. This sy ...

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Profiling DNA Methylation from Small Amounts of Genomic DNA Starting Material: Efficient Sodium Bisulfite Conversion and Subsequent Whole-Genome Ampli

noindent Sodium bisulfite modification-based fine mapping of methylated cytosines represents the gold standard technique for DNA methylation studies. A major problem with this approach, however, is that it results in considerable DNA degradation, and large quantities of genom ...

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Melting Curve Assays for DNA Methylation Analysis

The ability of sodium bisulfite to modify cytosines in a methylation-dependent manner allows the conservation of DNA methylation information during PCR amplification. PCR products amplified from bisulfite-modified DNA have significantly different base compositions ac ...

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Automated Fluorescent DNA Sequencing on the ABI PRISM 377

Fluorescent automated DNA sequencing based on ″one-lane, four-dye″ technology has played a pivotal role in the success of the human genome project. One Applied Biosystems 377 sequencer can produce at least two runs per day of sequence reads averaging 750 bases in length, each run produces up to 96 re ...

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Automated Fluorescent DNA Sequencing on the ABI PRISM 310 Genetic Analyzer

Three fundamental technologies have emerged in genetic analysis that have widespread and immediate benefits. DNA synthesis, fluorescence-based DNA analysis, and the polymerase chain reaction (PCR) are being integrated for a range of applications, from forensics to the understa ...

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Fluorescent Sequencing Protocols for the ALF

The development of polymerase chain reaction (PCR) (1), fluorescent DNA labels, and automated detection systems have revolutionized the efficiency, safety, and speed of DNA sequencing as compared to the original method of Sanger et al. (2), who published their chain terminating inhibit ...

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Exonuclease III Footprinting on Immobilized DNA Templates

DNA footprinting is a widely used method to locate the binding sites of protein on the DNA. It is based on the observation that a protein bound to DNA protects it from degradation by an enzyme or chemical reagent. Exonuclease III is a suitable probe to analyze the boundaries of a protein when it is necessary to el ...

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Surface Plasmon Resonance Assays of DNAProtein Interactions

Assaying sequence-specific DNA–protein complex formation in vitro often involves the use of specific labelling or modification of the components of the complex to provide unique signals that can be used to assess the affinity of the interaction. Surface plasmon resonance (SPR) spectr ...

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Defining the Thermodynamics of Protein/DNA Complexes and Their Components Using Micro-calorimetry

Understanding the forces driving formation of protein/DNA complexes requires measurement of the Gibbs energy of association, ΔG, and its component enthalpic, ΔH, and entropic, ΔS, contributions. Isothermal titration calorimetry provides the enthalpy (heat) of the binding reac ...

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Circular Dichroism for the Analysis of ProteinDNA Interactions

Circular dichroism (CD) is a well-established technique for the analysis of both protein and DNA structure. The analysis of protein–nucleic acid complexes presents greater challenges, but at wavelengths above 250 nm, the circular dichroism signal from the DNA predominates. Examples ...

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Fluorescence Spectroscopy and Anisotropy in the Analysis of DNAProtein Interactions

Fluorescence spectroscopy can be used as a sensitive non-destructive technique for the characterisation of protein–DNA interactions. A comparison of the intrinsic emission spectra obtained for a protein–DNA complex and for free protein can be informative about the environment of ...

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Analysis of Distant Communication on Defined Chromatin Templates In Vitro

Regulation of many biological processes in eukaryotes involves distant communication between the regulatory DNA sequences (e.g., enhancers) and their targets over the DNA regions organized in chromatin. However previously developed methods for analysis of communication in c ...

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Hydroxyl Radical Footprinting of ProteinDNA Complexes

This unit details the use of hydroxyl radicals to characterize protein–DNA interactions. This method may be used to assess the exact location of contacts between a protein and its cognate DNA and details of the complex structure. We describe several methods to prepare DNA templates for footpr ...

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Uranyl Photofootprinting

The uranyl-(VI) cation (UO2 2+) forms strong complexes with accessible phosphates of nucleic acid (DNA and RNA) backbones. Upon excitation with long wavelength ultraviolet light (λ = 300–420 nm), uranyl ions bound to backbone phosphates oxidize proximal sugars and induce nucleic acid back ...

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Ethylation Interference Footprinting of DNAProtein Complexes

Structural studies of DNA–protein complexes reveal networks of contacts between proteins and the phosphates, sugars and bases of DNA. A range of biochemical methods, termed chemical footprinting, aim to determine the functional groups on DNA which are protected in solution by bound pro ...

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Identification of Protein/DNA Contacts with Dimethyl Sulfate: Methylation Protection and Methylation Interference

Sequence-specific protein/DNA contacts direct most transcription factors to binding sites within the promoters of genes they regulate. Several chemical probes, such as dimethyl sulfate, have been used to obtain information on these sites of interaction. Protection and interfer ...

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Transgenesis in Drosophila melanogaster

Transgenesis in Drosophila melanogaster relies upon direct microinjection of embryos and subsequent crossing of surviving adults. The necessity of crossing single flies to screen for transgenic events limits the range of useful transgenesis techniques to those that have a very hi ...

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Transgenesis in Caenorhabditis elegans

Two efficient strategies have been developed and are widely used for the genetic transformation of the nematode Caenorhabditis elegans, DNA microinjection, and DNA-coated microparticle bombardment. Both methodologies facilitate the delivery of exogenous DNA into the devel ...

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