variety Protocols

Category: Plant Biology Protocols: Plant Genetics Protocols

AFLP was designed as a highly sensitive method for DNA fingerprinting to be used in a variety of fields. We are using this technology to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been identified genetically by mutant phenotype. Protocol includes: Generate polymorphic recombinant F2 (or F3) population; Isolate genomic DNA; Restriction of DNA; Ligation of adapters; Pre-amplification of template DNA; AFLP-PCR; etc. - [Read AFLP For Positional Cloning]

Category: Genetics and Genomics: Cancer Genetics Protocols

SKY has also been applied for the mouse genome, enabling investigators to extrapolate information from mouse models of cancer to their human counterparts. This review will address the advances that SKY has facilitated in the field of cancer cytogenetics, as well as its variety of application in the cancer research laboratories. - [Read Applications of SKY in Cancer Cytogenetics Review]

Category: Protein Protocols: Protein Extraction From Cells

Cell Lysate Extracts. Great protocols for cell lysis preparation from a variety of cell types. There are numerous methods of cell stimulation and lysis. For a given protein, Upstate’s Laboratories determine the specific treatment upon initial testing of its products. It is important to select the correct cell line, stimulation procedure (if any), and lysis protocol. Upstate. - [Read Cell Lysate Extracts]

Category: Transfection Protocols: Stable Transfection Protocols

Pulsed electrical fields can be used to introduce DNA into a wide variety of animal cells. Electroporation works well with cell lines that are refractive to other techniques, such as calcium phosphate-DNA coprecipitation. But, as with other transfection methods, the optimal conditions for electroporating DNA into untested cell lines must be determined experimentally. - [Read DNA Transfection by Electroporation]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Immunofluorescence Staining Protocols

Fluorescent dyes absorb light at certain wavelengths and in turn emit their fluorescence energy at a higher wavelength. Each dye has a distinct emission spectrum, which can be exploited for multicolor analysis. eBioscience antibodies are available conjugated to a wide variety of fluorochromes. - [Read Fluorescent Dyes for Flow Cytometric Analysis]

Category: Antibody Protocols: Antibody Labeling Protocols

This protocol describes the covalent coupling of antibodies to biotin. Biotin groups bind with extremely high affinity to streptavidin and avidin, both of which are available commercially coupled with enzymes, fluorescent dyes, or iodine. A biotinylated primary antibody, therefore, can be detected with any of a wide variety of different labels. The biotinylation reaction is simple and mild, and rarely inactivates the antibody. - [Read Labeling Antibodies with Biotin Protocol]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, etc. Protocol provides a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1–1.5 h. - [Read Laser-capture Microdissection Protocol]

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

The yeast, Saccharyomyces cerevisiae, has become an important organism in molecular, biochemical, and genetic analysis. The organism has specific requirements for growth under a variety of conditions. The media, both liquid and solid, simple, define, and complex are describe in this unit. Also included are methods for handling, storing, and shipping stock of yeast. - [Read Media and Culture of Yeast Protocol]

Category: DNA Microarray Protocols: DNA Methylation Microarray CpG

Kimura et al., NAR 2005. "easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylatio - [Read Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorec]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for northern hybridization. Protocol describes how to carry out northern hybridization at high stringency in phosphate-SDS-buffers.  Although a wide variety of formats are available, hybridization is usually performed in heat-sealable bags, roller bottles, or plastic boxes, as described here. - [Read Northern Hybridization Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Protocol for PCR genotyping from tail DNA. This protocol works well for a variety of genes and primer pairs including Tg and KO alleles. Oligonucleotide melting temperatures between 60° and 65° seem to work well. - [Read PCR Genotyping from Tail DNA Protocol]

Category: Microinjection Protocols: Microinjection Equipment

This protocol contains methods for pulling microinjection needles using two different models of pipette pullers. The advantage of pulling needles in the laboratory is that a variety of different needle types can be pulled, depending on the samples and cells being injected. An added advantage is cost; once a pipette puller has been purchased, boxes of glass capillaries are inexpensive compared to premade microinjection needles. - [Read Preparation (Pulling) of Needles for Gene Delivery by Microinjection Protocol]

Category: Cell Biology and Cell Culture: Endothelial Cell Protocols

Endothelial cells, which line blood vessels, can be prepared from a variety of tissues. They are frequently prepared from the umbilical vein, which is relatively easy to obtain. The procedure is clearly described and provides a large population of highly purified endothelial cells. There are also methods for obtaining endothelial cells from other tissues such as fat, skin, and mucosa. These methods require special care and generate smaller populations of cells. - [Read Preparation of Endothelial Cells Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

This protocol uses a "light mitochondrial" pellet from a mammalian liver homogenate. The gradient thus has to resolve a variety of denser components (peroxisomes, lysosomes, mitochondria) from the Golgi membranes, which have a low density in iodixanol (1.06-1.09 g/ml) [1]. The protocol is specifically tailored to the purification of Golgi membranes from this pellet and is unsuitable for the isolation or analysis of other organelles present in the light mitochondrial fraction. - [Read Purification of Golgi Membranes from a Light Mitochondrial Fraction in a Self-Generated Gradient]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

This protocol is not phenol-based, but does require the addition of chloroform. The Concert Plant Reagent is intended for the isolation of RNA from a wide variety of plant tissues including blue spruce needles, potato tuber etc. - [Read Purification of RNA from Plant Tissue Using the Concert Plant Reagent]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for southern hybridization of radiolabeled probes to nucleic acids immobilized on membranes. Protocol describes how to carry out Southern hybridizations at high stringency in phosphate-SDS buffers.  Although a wide variety of formats are available, most Southern hybridizations are carried out in heat-sealable bags, roller bottles, or plastic boxes. - [Read Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Small ubiquitin-like modifier (SUMO) is a small molecule, but has a variety of regulatory functions in cells. SUMO modification is involved in transcriptional regulation, subcellular localization, and protein-protein interactions. SUMO conjugation requires sequential E1-dependent activation, E2-dependent conjugation, and E3-dependent ligation steps. Protocol includes: In vivo and in vitro SUMOylation assay and deSUMOylation assay. - [Read Sumoylation and Desumoylation Assays for a Chromatin-Remodelling Complex In Vivo and In Vitro]

Category: Cell Biology and Cell Culture

Telomere length can vary from strain to strain and is sensitive to a variety of mutants affecting DNA and chromosome function. Includes: Gel Protocol; Southern blot using oligo probe. - [Read Telomere Length Determination Protocol]