found Protocols

Category: Xenopus Protocols: Xenopus Egg Extracts Protocols

Protocol for cytostatic factor extract preparation for spindle assembly. The quality of the eggs is essential for good CSF extracts. Always sacrifice quantity for quality when trying to make functional extracts. Discard any batches of eggs that have "puff balls" or activated eggs that constitute more than 10% of the eggs. Use laid eggs and collect eggs at about 16 to 17 hours after priming with Progesterone (found in Pregnant Mare Serum Gonadotropin (PMSG)). - [Read Cytostatic Factor (CSF) Extract Preparation for Xenopus Spindle Assembly Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

EMS is used at concentrations that induce multiple point mutations in each plant, such that mutant alleles of a specific locus are found at a rate of ~1 in 2000-5000 M2 plants. This high rate of mutagenesis makes possible the screening of relatively few plants to find those with the phenotype of interest, a particular advantage if the screen is laborious or if only a small number of genes mutate to the required phenotype. - [Read EMS Mutagenesis of Arabidopsis Seed Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Forward genetics is used to identify genes that are involved in particular biological processes. For example, genes required for disease resistance can be found by identifying mutants with reduced or increased disease resistance, genes that control flower development can be identified by searching for mutants with altered flower morphology, and genes encoding enzymes for tryptophan biosynthesis can be identified by searching for mutants that require exogenous tryptophan for growth. - [Read Forward Genetics in Arabidopsis: Finding Mutations that Cause Particular Phenotypes Protocol]

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

FuGene Transfection. They use 4ul, we found we could use as little as 1.5 ul of Fugene-6. Lamond Lab. - [Read FuGene Transfection]

Category: Immunology: Mononuclear Cell Isolation Protocols

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations (protocol may be found at www.methods.info). Reduction of the amount of microbeads in comparison to Miltenyi protocol reduces the costs of the experiment. - [Read Isolation of Monocytes from Enriched PBMCs using CD14 Magnetic Beads Protocol]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Confocal laser scanning microscopy (CLSM) is a relatively new light microscopical imaging technique which has found wide applications in the biological sciences. The primary value of the CLSM to the biologist is its ability to produce optical sections through a 3-D specimen-e.g., an entire cell or a piece of tissue - that, to a good approximation, contain information from only one focal plane. Article includes principle and applications of confocal laser scanning microscope. - [Read Looking Inside Cells and Tissues by Optical Sectioning with a Confocal Laser Scanning Microscope]

Category: Immunology

A method for ameliorating autoimmune disease by passive transfer of IVIg-primed leukocytes. Developed a method for ‘priming’ specific leukocyte populations with IVIg to study their potential role in ameliorating ITP upon passive transfer, which we describe herein. Using this technique, found that splenic CD11c+ dendritic cells reacted with IVIg, or several IVIg mimetic regimes, were primed to ameliorate ITP upon transfer to thrombocytopenic mice. - [Read Method for Ameliorating Autoimmune Disease by Passive Transfer of IVIg-Primed Leukocytes]

Category: Cytoskeleton Protocols: Microtubules Protocols

Negative staining is a rapid, qualitative method for analyzing microtubule structure at the EM level. Because negative staining involves deposition of heavy atom stains, structural artifacts such as flattening of the cylindrical microtubule and opening up of microtubules into flat sheets are common. Negative staining is very useful because of its ease, rapidity and lack of requirement for specialized equipment other than that found in a regular EM facility. - [Read Negative Stain Electron Microscopy of Microtubules Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

One step extraction for isolation of plant DNA. DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm2 in less than 20 min & no tube changes. Method was tested on several plant species. Method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated using standard DNA isolation. - [Read One-Step Isolation of Plant DNA Suitable for PCR Amplification]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols

Optimized Welsh DDRT-PCR Protocol. Provided by Mirta Grifman and found in Neuroscience Protocols (Nov 1995). - [Read Optimized Welsh DDRT-PCR Protocol]

Category: PCR Protocols

Optimized Welsh DDRT-PCR Protocol. Provided by Mirta Grifman and found in Neuroscience Protocols (Nov 1995). - [Read Optimized Welsh DDRT-PCR Protocol]

Category: Plant Biology Protocols: Photosynthesis and Respiration Protocols

The physiological reactions of mitochondria and chloroplasts can be reduced to a series of electron transfers, catalyzed by specific enzymes found within the organelles. Thus, we can study the component processes of photosynthesis and respiration by isolating the organelles and measuring specific enzyme activity associated with that organelle. - [Read Photosynthesis and Respiration - Introduction]

Category: Plant Biology Protocols: Arabidopsis Transformation Protocols

Protocol for a simplified Arabidopsis transformation. Found that the MS salts, hormone, etc. make no difference, that OD of bacteria doesn't make much of a difference, that vacuum doesn't even make much of a difference as long as you have a decent amount of surfactant present. Plant health is still a major factor - healthy fecund plants make a big difference! With this method you should be able to achieve transformation rates above 1%. - [Read Simple Arabidopsis Transformation Protocol]

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

The principle behind the pressure cooker method described here is to use extended periods of heat to break some of the subcellular structures that block antibody access. This approach is appropriate for handling specimens on glass slides. The major advantages of the pressure cooker method are the ability to handle a large number of slides simultaneously, the convenience of using metal racks, and the avoidance of any hot spots that are found in the microwave. - [Read Unmasking Hidden Epitopes Using the Pressure Cooker Protocol]