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丁香实验推荐阅读
Ribosome Display for Rapid Protein Evolution by Consecutive Rounds of Mutation and Selection

Directed evolution experiments are performed to improve the properties of proteins by creating a library of mutated genes of interest and selecting those genes that encode proteins exhibiting desired properties. Here, we present one of the methods to carry out an evolutionary experime ...

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Directed In Vitro Evolution of Reporter Genes Based on Semi-Rational Design and High-Throughput Screening

Marker genes, such as gusA, lacZ, and gfp, have been applied comprehensively in biological studies. Directed in vitro evolution provides a powerful tool for modifying genes and for studying gene structure, expression, and function. Here, we describe a strategy for directed in vitro evoluti ...

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Hot Start PCR

Hot Start activation approaches are increasingly being used to improve the performance of PCR. Since the inception of Hot Start as a means of blocking DNA polymerase extension at lower temperatures, a number of approaches have been developed that target the essential reaction components s ...

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Primer Design for RT-PCR

Primer design is a crucial initial step in any experiment utilizing PCR to target and amplify a known nucleotide sequence of interest. Properly designed primers will increase PCR amplification efficiency as well as isolate the targeted sequence of interest with higher specificity. Many ...

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Reverse Transcription of the Ribonucleic Acid: The First Step in RT-PCR Assay

Reverse transcription (RT) is the synthesis of complementary deoxyribonucleic acids (DNA) from single-stranded ribonucleic acid (RNA) templates. This process is catalyzed by the reverse transcriptase enzyme, which is the replicating enzyme of retroviruses. Reverse transc ...

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Skeletal Muscle RNA Extraction in Preparation for RT-PCR

Extraction of high quality RNA is paramount to successful RT-PCR, and here, a method proven optimal for skeletal muscle is described. While this method described is for use with skeletal muscle, it could be suitable for other types of mammalian tissue also. This method describes an approach to extr ...

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The Renaissance of Competitive PCR as an Accurate Tool for Precise Nucleic Acid Quantification

Here, we report a detailed procedure for the exact quantification of minute amounts of nucleic acids by competitive PCR. This technique entails the co-amplification of a target DNA or cDNA in a biological sample together with a known quantity of a target-specific standard, the competitor, whi ...

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Detection of Influenza A Virus Neuraminidase and PB2 Gene Segments by One Step Reverse Transcription Polymerase Chain Reaction

We describe a single step reverse transcription polymerase chain reaction protocol that can be used to amplify part of the neuraminidase gene segment (segment 6) from all nine subtypes of influenza A virus. The method has also been applied to amplify gene segment 1 of influenza A, which encodes the b ...

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Detection and Identification of CD46 Splicing Isoforms by Nested RT-PCR

CD46 (Membrane Cofactor Protein, MCP) is a transmembrane glycoprotein, which is expressed by all nucleated human cells whose purpose is to protect against autologous complement attack. In addition, CD46 can serve as a receptor for several viruses and bacteria and as a potent regulator of the i ...

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Detection of West Nile Viral RNA from Field-Collected Mosquitoes in Tropical Regions by Conventional and Real-Time RT-PCR

West Nile virus (WNV) is an emerging mosquito-borne flavivirus, which has rapidly spread and is currently widely distributed. Therefore, efforts for WNV early detection and ecological surveillance of this disease agent have been increased around the world. Although virus isolation is ...

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Simultaneous Detection of Bluetongue Virus RNA, Internal Control GAPDH mRNA, and External Control Synthetic RNA by Multiplex Real-Time PCR

Bluetongue is an insect-borne disease of domestic and wild ruminants that requires strict monitoring by sensitive, reproducible and robust methods. Real-time reverse transcription polymerase chain reaction (RT-qPCR) analysis has become the method of choice for routine viral di ...

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Detection of Antisense RNA Transcripts by Strand-Specific RT-PCR

Comprehensive genome annotation requires extensive cDNA analysis. This analysis has identified natural antisense transcripts (NATs), which are distinct from the microRNAs, siRNAs, and piRNAs, in a number of diverse eukaryotes. This wide conservation supports the possibility ...

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RT-LATE-PCR for mRNA and Viral RNA Detection and Quantification

LATE-PCR is an optimized form of asymmetric PCR that efficiently generates high levels of single-stranded DNA amplicons. Single-stranded amplicons are advantageous because, as shown in this chapter, they can be probed at low temperature(s) with one or more probes. Based on its properties, L ...

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Changes in Gene Expression of Caveolin-1 After Inflammatory Pain Using Quantitative Real-Time PCR

This chapter will take the reader through the steps involved in obtaining a gene expression profile using real-time PCR. Real-time PCR is an end-point measure of changes in gene expression that have occurred after a physiological event. It specifically describes the process by which real-ti ...

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Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction

The real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) has become the method of choice for the quantification of specific mRNAs. This method is fast, extremely sensitive, and accurate, requires only very small amounts of input RNA, and is relatively simple ...

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The Use of Comparative Quantitative RT-PCR to Investigate the Effect of Cysteine Incubation on GPx1 Expression in Freshly Isolated Cardiomyocytes

Intracellular cysteine availability is one of the major rate limiting factors that regulate the synthesis of the major antioxidant, glutathione. Little is known, however, about the effect of cysteine upon glutathione-associated enzymes in isolated heart cells. Such knowledge is im ...

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Real-time RT-PCR for Automated Detection of HIV-1 RNA During Blood Donor Screening

Real-time RT-PCR has become the method of choice for automated detection of viral RNA target sequences in the clinical laboratory. Besides commercially available certified test systems, a variety of so-called in-house methods have been described in the literature. Generally, approp ...

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A Method for Rapid Genetic Integration into Plasmodium falciparum Utilizing Mycobacteriophage Bxb1 Integrase

Genetic manipulation of the human malaria parasite Plasmodium falciparum has presented substantial challenges for research efforts aimed at better understanding the complex biology of this highly virulent organism. The development of methods to perform gene disruption, all ...

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Substrate-Induced Gene Expression Screening: A Method for High-Throughput Screening of Metagenome Libraries

The SIGEX (substrate-induced gene expression) method is a novel approach for the screening of gene (genome) libraries. In addition to the commonly used function- and sequence-driven approaches to screening, SIGEX provides a third option; in SIGEX, positives are identified using a repor ...

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Screens for Active and Stereoselective Hydrolytic Enzymes

A procedure for the high-throughput screening (HTS) of esterases is described. This includes a pretest for discrimination of active and inactive clones using an agar plate overlay assay, the enzyme expression in microtiter plates and the measurement of activity and enantioselectivi ...

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