yields Protocols

Category: Drosophila Protocols

Enzyme-linked reagents give excellent sensitivity and use a simple light microscope for detection. A range of enzymes is available, but for staining in situ, horseradish peroxidase will suit most needs. Diaminobenzidine (DAB) is one of the most sensitive substrates for horseradish peroxidase. It yields an intense brown product that is insoluble in both water and alcohol. It can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. - [Read Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents Protocol]

Category: Plant Biology Protocols: Arabidopsis Protein Peptide Protocols

The simplest way to analyze proteins is in unfractionated extracts. However, it is often desirable to fractionate proteins, e.g, by size. This procedure extracts total protein from Arabidopsis samples. Typical yields are ~2-3 mg/ml (using rosette leaves) or 6-8 mg/ml (using young seedlings). - [Read Extraction of Total Protein from Arabidopsis Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

Protocol describes the purification of mouse T cells, B cells, and T cell subsets using magnetic bead separation. Isolation of cell subsets using magnetic beads is quick, simple, and reliable and can result in high yields of very pure cells. - [Read Fractionation of T and B Cells Using Magnetic Beads Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocol describes the purification of mouse T cells, B cells, and T cell subsets using magnetic bead separation. Isolation of cell subsets using magnetic beads is quick, simple, and reliable and can result in high yields of very pure cells. - [Read Fractionation of T and B Cells Using Magnetic Beads Protocol]

Category: Competent Cells Protocols

This is a very easy protocol which yields transformation efficiencies appropriate for routine cloning and transformation. - [Read Frozen Competent E. coli Preparation and Transformation Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This procedure describes the isolation and culture of adult mouse cardiac myocytes from one heart. The isolation routinely yields approximately 1 million rodshaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from One Heart Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol describes the isolation and culture of adult mouse cardiac myocytes from one heart. The isolation routinely yields approximately 1 million rodshaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from One Heart Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This procedure describes the isolation and culture of adult mouse cardiac myocytes from two or more hearts. Includes modifications for the digestion of two or more hearts in the same procedure and subsequent pooling of myocytes derived from the multiple hearts. The isolation procedure is performed by one or more technicians and routinely yields approximately 1 million rod-shaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from Two or More Hearts Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

Protocol for isolation of DNA from mammalian cells grown in 96-well microtiter plates. Each well yields sufficient genomic DNA for several standard PCRs or for analysis in a single lane of a Southern hybridization. - [Read Isolation of DNA from Mammalian Cells Grown in 96-well Microtiter Plates Protocol]

Category: Mouse Protocols

Provides two protocols that may be used to isolate IHL. One can be used to isolate IHL from multiple livers in parallel, whereas the more elaborate alternate protocol yields more cells per liver but is more appropriately used to recover the IHL from a single liver. - [Read Isolation of Mouse Intrahepatic Lymphocytes Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

Collagenase perfusion of rat liver yields a hepatocyte suspension which may be exposed to test compounds in order to assess their effects on cell viability and enzyme leakage. - [Read Isolation of Rat Hepatocytes Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol fopr markers of pulsed-field gel electrophoresis. Markers for pulsed-field gel electrophorsis can be generated by ligation of linear monomers of bacteriophage {lambda} DNA (48.5 kb) into a nested series of concatemers.  This procedure yields a series of concatemers that contain up to 20 tandemly arranged copies of bacteriophage DNA. - [Read Markers for Pulsed-field Gel Electrophoresis Protocol]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The number of assay steps has been minimized as much as possible to expedite sample processing. The MTT Reagent yields low background absorbance values in the absence of cells. Includes: Determining optimal cell counts, performing an assay, data interpretations and troubleshooting. - [Read MTT Cell Proliferation Assay Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Many replacement vectors (e.g., the EMBL series, {lambda}2001, and {lambda}DASH) contain a series of restriction sites, arranged in opposite orientations, at each end of the central stuffer fragment. Digestion of these vectors with two different restriction enzymes yields left and right arms, a stuffer fragment, and short segments of the polycloning sites. These can easily be removed from the arms by differential precipitation with isopropanol or spun-column chromatography. - [Read Preparation of Bacteriophage lambda DNA Cleaved with Two Restriction Enzymes Protocol]

Category: Cytogenetics Protocols

An ideal method of tissue preparation ensures both good specimen morphology and that the target molecules are in the optimum state for probe access and hybridization. DNA:DNA in situ hybridization is usually carried out on chromosome spread preparations where chromosome and nuclei are released from cells and spread on a glass microscope slide. This method yields well separated and enlarged chromosomes with good morphology which can be analyzed in transmitted light or fluorescence microscopes. - [Read Preparation of Chromosome Spreads]

Category: Mouse Protocols: Mouse Genetics Protocols

Method is used chiefly to genotype transgenic and knockout mice. Each 6-10-mm snippet of mouse tail yields 50-100 µg of DNA that can be used in dot or slot blotting to detect a transgene of interest, in Southern hybridization to detect DNA fragments that are <20 kb in size, and as a template in PCRs. - [Read Preparation of Genomic DNA from Mouse Tails and Other Small Samples Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

Simple protocol is used to extract DNA from small numbers of cultured cells and from fragments of soft or bony tissues.  The method is used chiefly to genotype transgenic and knockout mice.  Each 6-10-mm snippet of mouse tail yields 50-100 µg of DNA that can be used in dot or slot blotting to detect a transgene of interest, in Southern hybridization to detect DNA fragments that are <20 kb in size, and as a template in PCRs. - [Read Preparation of Genomic DNA from Mouse Tails and Other Small Samples Protocol]

Category: DNA Protocols: DNA Extraction Protocols: Agarose Gel DNA Extraction Protocols

Rapid Elution of DNA Agarose Gels Protocol.  This method allows quick purification of DNA fragments from agarose gels for use in cloning and other reactions. Higher yields of purified DNA can be obtained from commercially available purification kits, however greater ligation efficiencies per given amount of DNA have been seen with the use of these described spin columns. Hahn Lab. - [Read Rapid Elution of DNA Agarose Gels Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Mammalian DNA prepared from blood or tissues as described in this protocol is 20-50 kb in size and suitable for use as a template in PCRs.  The yields of DNA vary between 0.5 and 3.0 µg/mg tissue or 5 and 15 µg per 300 µl of whole blood. - [Read Rapid Isolation of Mammalian DNA Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

Recovery of bands of DNA from agarose gels by electrophoresis onto a sliver of DEAE-cellulose membrane can be performed simultaneously on many samples and reliably gives high yields of fragments between 500 bp and 5 kb in length. - [Read Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-cellulose Membranes Protocol]

Category: Fungi Protocols: Neurospora Protocols

DNA isolation method yields an average of 0.6 micrograms of genomic DNA that is suitable for Southern analysis or PCR. Starting with fresh mycelium, 20 to 40 samples can be processed in approximately two hours. Better yields (about 5 micrograms) may be obtained by suspending approximately 100 microliters of ground lyophilized mycelium in 500 microliters of isolation buffer and following the protocol. - [Read Small Scale DNA Preps for Neurospora crassa Protocol]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast DNA Isolation Protocols

This method reproducibly yields several micrograms of yeast DNA that can be efficiently cleaved by restriction enzymes and used as a template in PCR. - [Read Small-scale Preparations of Yeast DNA Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Technique yields a heterogeneous population of short radiolabeled molecules 200-300 nucleotides in length. These probes are synthesized, as in Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates, by extension of an oligonucleotide primer on a single-stranded DNA template. The radiolabeled products of the reaction are then separated from the template by electrophoresis through a denaturing gel from which they are eluted directly into hybridization buffer. - [Read Synthesis of Single-stranded DNA Probes of Heterogeneous Length from Bacteriophage M13 Templates]

Category: Viral Manipulation Protocols: Phage Protocols

Protocol describes methods for recovery and purification of recombinant clones of bacteriophage P1 or PAC DNAs from bacteria. Because of their large size, these DNAs are sensitive to shearing forces and must be handled carefully. This protocol generally yields P1 DNA that works well as a substrate or template in enzymatic reactions. - [Read Working with Bacteriophage P1 and Its Cloning Systems Protocol]