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丁香实验推荐阅读
Use of Natural and Artificial Chromosome Vectors for Animal Transgenesis

The ability to use hCFs as vectors for introducing large stretches of human DNA into mice was first demonstrated in 1997 (1). Transferred hCFs were stably maintained as an extra chromosome in the somatic cells of mice, and the human genes included in them were expressed under proper tissue-specific ...

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Plasmid Vectors for Marker-Free Chromosomal Insertion of Genetic Material in Escherichia coli

Amethod to achieve the insertion of genetic material into the chromosome of Escherichia coli is described. The method is based on the use of integration vectors from the pBRINTs-rAnbR family. These vectors offer the choice of using the antibiotics chloramphenicol, gentamycin, or kanamy ...

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Pyrosequencing-Based Strategies for Improved Allele Typing of Human Leukocyte Antigen Loci

Successful transplantation of tissue during solid organ and bone marrow transplantation relies on accurate determination of the human leukocyte antigen (HLA) phenotype of the potential donor(s) and recipient. Matching donor with recipient for a kidney transplant generally mea ...

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Pyrosequencing of Phage Display Libraries for the Identification of Cell-Specific Targeting Ligands

High combinatorial phage display libraries have become an important tool in the search for ligand-receptor interactions. The advantage this approach offers is the ability to screen large repertoires of peptides, displayed on the coat proteins of bacteriophages, against a target at the ...

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Gene Copy Number Detection in Animal Studies

Sensitive methods for the quantification of DNA fragments can be used to study an individual’s genetic constitution for duplicated regions of the genome or to determine the relative proportion of different DNA fragments in heterogeneous samples such as pooled DNA from different indiv ...

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Detection of Allelic Imbalance in Gene Expression Using Pyrosequencing

Single-nucleotide polymorphisms (SNPs) are common in the human genome, with more than 11 million SNPs having frequencies greater than 1%. The challenge is to identify the minority of functional SNPs from the large number of SNPs that are expected to be silent. Whereas coding variants are unusua ...

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Statistical Comparison of Two or More SAGE Libraries: One Tag at A Time

Several statistical tests have been introduced for the comparison of serial analysis of gene expression (SAGE) libraries to quantitatively analyze the differential expression of genes. As each SAGE library is only one measurement, the necessary information on biological variat ...

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Duplicate Ditag Analysis in LongSAGE

The Long serial analysis of gene expression (SAGE) protocol generates ditags from tags with overlapping overhangs, thereby increasing the probability of duplicate ditag formation in LongSAGE. In this chapter, a tool is presented that facilitates the analysis of duplicate ditags in Lo ...

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Single-Nucleotide Polymorphism Mapping

Single-nucleotide polymorphism (SNP) mapping is the easiest and most reliable way to map genes in Caenorhabditis elegans. SNPs are extremely dense and usually have no associated phenotype, making them ideal markers for mapping. SNP mapping has three steps. First, recombinant mutant an ...

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C. elegans Deletion Mutant Screening

The methods used by the Caenorhabditis elegans Gene Knockout Consortium are conceptually simple. One does a chemical mutagenesis of wild-type C. elegans, and then screens the progeny of the mutagenized animals, in small mixed groups, using polymerase chain reaction (PCR) to identify pop ...

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Techniques for Analysis, Sorting, and Dispensing of C. elegans on the COPAS Flow-Sorting System

The COPAS™ Biosorter is a flow cytometer designed to accommodate large objects the size of Caenorhabditis elegans. This instrumentation brings high-speed automated analysis and sorting to this small model organism. The Biosort system optically analyzes and sorts living multicel ...

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Automated Imaging of C. elegans Behavior

Automated systems for recording and analyzing behavior have many applications for the study of neurobiology in Caenorhabditis elegans. In particular, machine-based approaches allow for precise quantitative definitions of behavioral phenotypes that have traditionally ...

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Functional Genomic Approaches in C. elegans

The nematode Caenorhabditis elegans is an extraordinarily powerful model organism for the application of functional genomic approaches. Two such approaches, whole genome microarray analysis and genome-wide RNA interference (RNAi)-mediated phenotypic screening, are hi ...

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Delivery Methods for RNA Interference in C. elegans

This chapter describes four methods for delivery of double-stranded RNA to Caenorhabditis elegans (injection, feeding, soaking, and in vivo delivery), and suggests schemes that should facilitate detection of specific gene silencing.

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Construction of Plasmids for RNA Interference and In Vitro Transcription of Double-Stranded RNA

Double-stranded RNA (dsRNA)-induced gene silencing in Caenorhabditis elegans involves the manufacture and delivery of defined sequences of dsRNA to the organism, followed by a careful monitoring for loss-of-function phenocopies in treated animals. In this chapter, we describe h ...

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An Overview of C. elegans Biology

The establishment of Caenorhabditis elegans as a “model organism” began with the efforts of Sydney Brenner in the early 1960s. Brenner’s focus was to find a suitable animal model in which the tools of genetic analysis could be used to define molecular mechanisms of development and nervous system ...

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High-Resolution, Genome-Wide Mapping of Chromatin Modifications by GMAT

One major postgenomic challenge is to characterize the epigenomes that control genome functions. The epigenomes are mainly defined by the specific association of nonhistone proteins with chromatin and the covalent modifications of chromatin, including DNA methylation and pos ...

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DeepSAGE: Higher Sensitivity and Multiplexing of Samples Using a Simpler Experimental Protocol

Combining serial analysis of gene expression (SAGE) with pyrophosphatase-based ultra-high-throughput DNA sequencing provides increased sensitivity and cost-effective gene expression profiling. The combined techniques obviate the formation and cloning of concate ...

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Low-Cost-Medium Throughput Sanger Dideoxy Sequencing

Serial analysis of gene expression (SAGE) requires the sequencing of DNA. The principal cost of SAGE is largely determined by the cost of sequencing. Therefore, it is important to have access to a robust and affordable sequencing system. Here, we describe such a system based on the sequencing of ampl ...

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SuperSAGE

As a tool for high-throughput, quantitative gene expression analysis, serial analysis of gene expression (SAGE) is one of the most powerful techniques. However, the short size of tags (14bp) has hindered the application of SAGE to a vast majority of eukaryotes without sufficient genomic res ...

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