produced Protocols

Category: PCR Protocols: AFLP PCR

Amplified Fragment Length Polymorphisms and Microsatellites: A phylogenetic perspective. Julian P. Robinson, Stephen A. Harris. What are AFLPs and how are they produced? How AFLPs have been used? Problems? Restriction Enzymes and Primers. AFLP Reproducib - [Read Amplified Fragment Length Polymorphisms and Microsatellites]

Category: Plant Biology Protocols: Plant Genetics Protocols

Virus-induced gene silencing (VIGS) uses a virus to deliver a sequence from a gene of interest into a host plant. The virus carrying the fragment of the gene of interest must be capable of replication if dsRNA is to be produced. One or two leaves are inoculated with Agrobacterium strains carrying the VIGS vector possessing the gene fragment. The virus then replicates and spreads throughout the plant, mediating silencing. - [Read Delivery of dsRNA into Plants by VIGS Methodology]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors.  This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. - [Read ELISA Method Measure Inhibition COX Enzymes]

Category: ELISA Protocols

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. - [Read ELISA Method to Measure Inhibition of the COX Enzymes Protocol]

Category: Transfection Protocols: Stable Transfection Protocols

CHO Lec3.2.8.1 cells have 4 independent mutations in the N and O glycosylation pathways. When cultured with alpha-glucosidase I inhibitor N-butyl-deoxynojirimycin, glycoproteins produced in CHO Lec3.2.8.1 cells are completely susceptible to Endo H digestion. Endo H cleaves chitobiose, leaving a single N-linked N-acetylglucosamine per site which is ideal for maintenance of protein solubility and special carb-protein interactions, such as between the first N-acetyl glucosamine residue and tryp. - [Read Establishment of Stable Transfectant of CHO Lec Cells Protocol]

Category: Cell Culture Protocols

Protocol describes a method for estimation of mammalian cell number in a defined volume of medium using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted. - [Read Estimation of Cell Number by Hemocytometry Counting Protocol]

Category: DNA Microarray Protocols

This protocol describes the steps required to produce a cDNA microarray. Gene-specific DNA is produced by PCR amplification of purified template plasmid DNAs from cloned ESTs. The PCR product is purified by ethanol precipitation, thoroughly resuspended in - [Read Fabrication Protocol for DNA Microarrays]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Sorting Protocols

Flow cytometry is a widely used method for characterizing and separating individual cells. This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules. Includes: Immunofluorescence Staining and Flow Cytometry Analysis. - [Read Flow Cytometry Analysis Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Flow cytometry is a widely used method for characterizing and separating individual cells. This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules. Includes: Immunofluorescence Staining; Flow Cytometry Analysis. - [Read Flow Cytometry Analysis Protocol]

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

This procedure describes a method for establishing short-term explant cultures of oesophageal mucosa. Adverse effects produced by exposure to radiation or test compounds can be detected as an inhibition of cell outgrowth. - [Read Human Oesophageal Culture Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

Protocol describes a method for establishing short-term explant cultures of oesophageal mucosa.  Adverse effects produced by exposure to radiation or test compounds can be detected as an inhibition of cell outgrowth. - [Read Human Oesophageal Culture Protocol]

Category: Chromatography Protocols: Gas Chromatography Protocols

Protocol describes monosaccharide analysis by methanolysis. The method gives more complex chromatograms than the corresponding alditol acetate method, because up to four methyl glycosides can be produced from each monosaccharide.  However, the method is easier to perform. - [Read Monosaccharide Analysis by Methanolysis 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: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

The method uses Trizol, a phenol-base reagent produced by Invitrogen. - [Read Preparation of RNA from Paraffin-Embedded Fixed Tissue Using Trizol Protocol]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

siRNAs produced upon the addition of dsRNA to Drosophila embryo extract are enriched in a micrococcus-nuclease-resistant fraction.  After proteinase K treatment and dephosphorylation with calf intestinal phosphatase, these siRNAs mediate efficient RNAi in vitro. - [Read Preparation of siRNAs from Drosophila Embryo Extracts Protocol]

Category: Avian Bird Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes. The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII). The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. After purification, the RNA strands are annealed to yield a dsRNA molecule suitable for RNAi in avian embryos. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes.  The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII).  The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

Protocol for purification of RNA from animal cells using Trizol. Trizol is a phenol-based reagent produced by Invitrogen. - [Read Purification of RNA from Animal Cells Using Trizol]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

The activity of ß-glucuronidase (GUS) can be accurately determined in intact plant tissue using 4-methylumbelliferyl ß-D-glucuronide (4-MUG) as a substrate. Upon hydrolysis by GUS, the fluorochrome 4-methyl umbelliferone (4-MU) is produced. This method is based on the permeability of both 4-MUG and 4-MU through plant tissue. It consists of incubation of the tissue with the reagent and quantification of the fluorescence emitted by 4-MU in the solution. GUS activity in each sample can be... - [Read Quantitative GUS Activity Assay in Intact Plant Tissue Protocol]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

Using excitation at 365 nm and measuring emission at 455 nm, the amount of 4-MU produced can be quantified. Under these conditions, background fluorescence from the substrate is negligible, especially if the appropriate filter is selected. - [Read Quantitative GUS Activity Assay of Plant Extracts]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this protocol, double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]

Category: PCR Protocols

Protocol describes how double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]