widely Protocols

Category: Protein Protocols: Protein Quantitation Concentration Protocols

Absorbance assays are fast and convenient, since no additional reagents or incubations are required.  No protein standard need be prepared.  The assay does not consume the protein.  The relationship of absorbance to protein concentration is linear.  Because different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures. - [Read Absorbance Assay 280 nm]

Category: DNA Protocols: DNA Sequencing Protocols

Protocol first describes the vector preparation and, then, describes the insert preparation.  Vital to have an excellent vector in order to produce a sequencing library.  Protocol employs the male-specific coliphage M13 as the sequencing vector.  M13 is a filamentous phage with a single-stranded, circular genome.  M13 is widely used as a vector because many versions are available commercially and because M13 has certain advantages. - [Read Construction of the Sequencing Library Protocol]

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Cryogenic preservation (storage below -100°C) of cell cultures is widely used to maintain backups or reserves of cells without the associated effort and expense of feeding and caring for them. The success of the freezing process depends on four critical areas: Proper handling and gentle harvesting of the cultures; Correct use of the cryoprotective agent; A controlled rate of freezing; Storage under proper cryogenic conditions. - [Read Cryogenic Preservation and Storage of Animal Cells Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

Microsatellite markers, also referred to as STMS (SequenceTagged Microsatellite Sites) or STR (Short Tandem Repeats) are widely used as molecular markers for intraspecific genotyping, molecular mapping and breeding purposes. The method described is an efficient,fast and relatively inexpensive way to obtain microsatellite markers without post-cloning selection methods. So far, the method has been successful in onion (Allium cepa L.), a plant with a large genome and for pathogenic fungi. - [Read Enrichment for Microsatellite Sequences in Onion (Allium cepa L.) Protocol]

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: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for fluorescence in situ hybridization (FISH) for DNA replication origins. Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique used for the detection of specific chromosomal rearrangements and applicable to many different specimen types. FISH is widely used for several diagnostic applications. - [Read Fluorescence In Situ Hybridization (FISH) for DNA Replication Origins Protocol]

Category: Cell Biology and Cell Culture: Hepatocyte Cell Culture

Primary culture of hepatocytes is an in vitro model widely used to ... protocols for cytochromes P450IIIA in vivo and in primary cultures of animal and ...Human Hepatocyte Culture Protocol - [Read Human Hepatocyte Culture Protocol]

Category: Immunohistochemistry Protocols

Protocol for immunohistochemistry on fixed, paraffin-embedded sections. This method is widely used and applies to the detection of the overwhelming majority of antigens, with few exceptions for which enzymatic retrieval is required. The method uses a strong chelating agent, EDTA. Includes: Double indirect AP; AP Developing solution; Indirect immunohistochemistry with avidin-biotin and HRP; HRP Developing solution. - [Read Immunohistochemistry on Fixed, Paraffin-Embedded Sections Protocol]

Category: Protein Protocols: Lowry Protein Assay

Lowry Protein Assay. The Lowry procedure is one of the most venerable and widely-used protein assays, being first described in 1951 [Lowry et al., J. Biol. Chem. 193: 265-275 (1951)]. Under alkaline conditions, copper complexes with protein. When folin phenol reagent (phospho-molybdic-phosphotungstic reagent) is added, the Folin-phenol reagent binds to the protein. Bound reagent is slowly reduced and changes color from yellow to blue. P.J. Hansen, Dept. of Animal Sciences, University of Florida. - [Read Lowry Protein Assay]

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

In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rRNA, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies. - [Read Protocol: Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

This protocol describes nuclear and cytoplasmic fractionation of tissue culture cells and a method for Western blot detection of proteins using the Odyssey Infrared Imaging System. This protocol was used to detect expression of the "small" Tap protein in 293T, HeLa and COS cells. The Odyssey system has several advantages over the more widely used chemiluminescent detection methods. - [Read Western Blot Analysis of Sub-Cellular Fractionated Samples Using the Odyssey Infrared Imaging System]