CSH protocols最新文献

INTRODUCTIONTomatoes can be easily grown in a field, in a greenhouse, or in a growth cabinet. They need acidic soil (pH 6.0-6.8), a lot of light, and water. The optimum temperature for growing tomato plants and fruit is 18°C-24°C. This protocol describes how to germinate tomato seeds, cultivate adult plants, and harvest seeds from fruit.

INTRODUCTIONThis protocol describes how to cross tomato plants. Crossing is important for the genetic analysis and breeding of tomatoes. Tomatoes are self-pollinating plants; thus, emasculation (removal of the anthers from the female parent) is essential. All wild tomato species can be crossed with cultivated tomatoes (although it may be difficult); this is useful because wild tomatoes are a great source of desirable traits. Most commercial tomatoes are F(1) hybrids, and the seeds for them were produced by crossing two parent tomatoes.

INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This approach, termed "Nucleic Acid Protein Programmable Array" (NAPPA), enables the simultaneous expression of proteins in microarray format without the need for individual protein purification. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes preparation of the bacterial cultures in deep-well blocks.

INTRODUCTIONIn this protocol, fertilized Astyanax mexicanus eggs are transferred into larger dishes to reduce crowding and are observed until they begin to hatch into fry.

INTRODUCTIONTransformation is an essential technique to analyze the function of genes. Tomato can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. This protocol describes an Agrobacterium-mediated transformation method for tomato.

INTRODUCTIONIn this protocol, we describe methods for cultivating Antirrhinum (snapdragon) species. These plants are easily grown, provided that they have sufficient light and are not overwatered. In good conditions, most species will flower and produce seeds within 3-4 mo. Strongly growing plants should suffer from few pests or diseases, but we also prescribe methods for dealing with microbes and insects that commonly damage Antirrhinum.

INTRODUCTIONIn this procedure, nematodes disembark from a beetle carcass and feed on Escherichia coli OP50. The nematodes are then monitored for a few days and identified using simple morphological characteristics. This method is rapid, easy, and biased for Pristionchus species.

INTRODUCTIONIn recent years, planarians have been increasingly recognized as an emerging model organism amenable to molecular genetic techniques aimed at understanding complex biological tasks commonly observed among metazoans. Growing evidence suggests that this model organism is uniquely poised to inform us about the mechanisms of tissue regeneration, stem cell regulation, tissue turnover, pharmacological action of diverse drugs, cancer, and aging. This article provides an overview of the planarian model system with special attention to the species Schmidtea mediterranea. Additionally, information is provided about the most popular use of this organism, together with modern genomic resources and technical approaches.

INTRODUCTIONBacteria growing in biofilms often express a different subset of genes compared to the same strains growing planktonically. Quantitative reverse transcriptase real time PCR (qRT-PCR) can be used effectively to quantify the number of RNA transcripts of specific genes from bacteria growing in biofilms. qRT-PCR has a large dynamic range and may be used to verify gene expression data obtained from microarrays. In addition, qRT-PCR is sensitive, and therefore may be used to quantify gene expression from biofilm samples where only a small amount of biological material is available, as in samples obtained by laser capture microdissection microscopy (LCMM). The most commonly used qRT-PCR methods are the SYBR Green and dual-labeled probe (Taqman) approaches. Both approaches use reverse transcription to convert mRNA to cDNA, followed by PCR amplification of the cDNA. This article describes steps involved in aspects of qRT-PCR including (1) primer design, (2) primer and probe performance testing, (3) qRT-PCR using the Corbett Rotor-Gene system, and (4) data export and analysis.

INTRODUCTIONThe Antirrhinum species group comprises approximately 20 morphologically diverse members that are able to form fertile hybrids. It includes the cultivated snapdragon Antirrhinum majus, which has been used as a model for biochemical and developmental genetics for more than 75 yr. The research infrastructure for A. majus, together with the interfertility of the species group, allows Antirrhinum to be used to examine the genetic basis for plant diversity.