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丁香实验推荐阅读
Use of Escherichiu coli (E. coli) lacZ (-Galactosidase) as a Reporter Gene

Our understanding of the molecular mechanisms that govern gene expression has been facilitated by the ability to introduce recombinant DNA molecules into heterologous cellular systems both in vitro and in vivo. One approach to defining DNA sequences important in the regulation of gene ...

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Application of the Firefly Luciferase Reporter Gene

Reporter genes have become powerful tools to study regulation of gene expression in eukaryotes. In particular, chimeric transcription units generated by the fusion of the appropriate DNA regulatory sequences to reporter genes have led to the identification of a great number of DNA contr ...

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Use of Vectors to Confer Resistance to Antibiotics G418 and Hygromycin in Stably Transfected Cell Lines

The development of dominant selection markers to identify eukaryotic cells that have undergone a gene transformation event has greatly facilitated molecular genetic studies in higher eukaryotic cells. Selection schemes based on resistance to antibiotic cytotoxicity (1,2) w ...

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Preparation of Recombinant Plasmid DNA for DNA-Mediated Gene Transfer

Recombinant plasmid constructs are frequently employed in transfection experiments. With the availability of a wide spectrum of specialized and versatile eucaryotic cloning/expression vectors, investigators have been given powerful tools to expedite the elucidation of ...

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Selection of Cells Defective in Pyrimidine (TK) and Purine (APRT and HPRT Salvage: Development of Host Strains Appropriate for Transfection

The usefulness of the purine and pyrimidine salvage pathways in the study of the mechanisms of mutation and in the selection of cell lines stably transformed by vectors expressing these genes is well documented. Unfortunately, many investigators are deterred from selecting new host stra ...

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Quantitative and Qualitative Analysis of Exogenous Gene Expression by the S1 Nuclease ProtectionAssay

The Sinuclease protection procedure allows the precise definition of the beginning and end of gene transcripts as well as the position of intron/ exon boundaries in a gene. Alternatively, when these parameters are already known, the technique can be used to quantify transcript levels in a vari ...

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Northern Blot Analysis of Gene Expression

In the analysis of gene expression, the steady-state level of RNA transcripts is one of the most convenient parameters used to monitor the activity of an endogenous or introduced gene in cell lines and tissues. A variety of methods, such as Sl hybridization (Chapter 21, this vol), RNase protection (C ...

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Primer Extension Analysis of mRNA Isolated from Transfected Cell Lines

Primer extension is a relatively quick and convenient means by which transcription from a gene transfected into tissue-culture cells can be monitored. The technique can be used to accurately determine the site of transcription initiation or to quantify the amount of cap-site-specific m ...

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Analysis of Transcriptional Initiation in Isolated Nuclei

The level of expression of a given gene in a particular cell type is reflected by the concentration of the resultant messenger RNA. This is subject to regulation during a number of processes, including synthesis, processing, export from nucleus to cytoplasm, and degradation. Clearly, the rate of ...

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Immunoperoxidase Staining of Gene Products in Cultured Cells Using Monoclonal Antibodies

Antibodies, in general, provide the most sensitive and specific methods for detecting the protein products of genes. Immunoperoxidase techniques described here detect 103-105 molecules/cell (depending on whether the protein is dispersed within the cell or concentrated at high d ...

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Constructing STR Multiplex Assays

Multiplex polymerase chain reaction (PCR) refers to the simultaneous amplification of multiple regions of deoxyribonucleic acid (DNA) using PCR. Commercial short tandem repeat (STR) assays that can coamplify as many as 16 different loci have become widely used in forensic DNA typing. T ...

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Y Chromosome STR Typing

Because of their unique transmission properties and male specificity, markers located on the nonrecombining region of the Y chromosome (NRY) have become an important tool in forensic investigation. In the past few years, more than 50 polymorphic Y chromosome-specific short tandem rep ...

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Using Online Databases for Developing SNP Markers of Forensic Interest

In this chapter we review and compare the online single nucleotide polymorphism databases that are now available as research tools. We give an outline of the search strategies that can be used to ensure the most appropriate loci for forensic applications are chosen.

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Genotyping SNPs With the LightCycler

Here, a single nucleotide polymorphism typing methodology is described based on polymerase chain reaction monitoring, in real time, of fluorescently labeled amplified products using the LightCycler. The main advantages of the system are the time required for the analysis (about 20 mi ...

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SNP Typing in Forensic Genetics: A Review

Single nucleotide polymorphisms (SNPs) are emerging as new markers of interest to the forensic community because of their abundance in the human genome, their low mutation rate, the opportunity they present of analyzing smaller fragments of deoxyribonucleic acid (DNA) than with short t ...

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New Rapid Multicolor PRINS Protocol

In the multiple-color primed in situ labeling (multi-PRINS) technique, using ddNTPs between two PRINS reactions can block the free 3’-end generated in the previous PRINS reaction, thus avoiding the next PRINS reaction, using it as a primer to perform spurious elongation at nondesired sites. ...

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PRINS Combined With Peptide Nucleic Acid Labeling

Both primed in situ labeling (PRINS) and peptide nucleic acid (PNA) technologies have emerged as research techniques, but they have quickly evolved to applications in biological diagnosis assays. The two procedures present several features (specificity, discriminating abili ...

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PRINS Combined With Indirect Immunofluorescence

Primed in situ labeling (PRINS) has proven to be an attractive alternative to fluorescence in situ hybridization for in situ DNA labeling and for being combined on the same slide with others methods. For the aim of a study on the asymmetrical segregation of the chromosomes of the megakaryocytes dur ...

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PRINS for the Detection of Unique Sequences

Primed in situ labeling (PRINS) can be used to localize single-copy genes and unique sequences. Using a modified PRINS method that incorporates multiple primers for the same sequence, single-step annealing and extension, anti-Taq DNA polymerase antibody, and stringent washing, we loc ...

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Single-Copy and Point Mutation Analysis (Rolling-Circle PRINS)

This protocol is a prototype for a series of upcoming procedures aimed at the detection of single molecules of nucleic acids in situ. It uses circular probes that, upon hybridization to their target molecule, can template rolling-circle DNA synthesis, if the target also provides a primer for ini ...

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