regions Protocols

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Method to identify genomic targets of DNA-binding factors. Chromatin immunoprecipitation (ChIP) assay on high-throughput microarray based methods for discovering genomic regions occupied by human DNA-binding proteins. Oberley and Farnham. - [Read ChIP on ChIP Protocol PDF]

Category: PCR Protocols: PCR Cloning Protocols

Pairs of oligonucleotide primers used in PCR are often designed with restriction sites in their 5' regions.  In many cases, the sites are different in the two primers.  In this case, amplification generates a target fragment whose termini now carry new restriction sites that can be used for directional cloning into plasmid vectors.  The purified fragment and the vector are digested with the appropriate restriction enzymes, ligated together, and transformed into E. coli. - [Read Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA Protocol]

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

Three Ambion kits were used to quantitate specific miRNAs and to detect differential miRNA expression in various mouse brain regions and cell types isolated by laser capture microdissection (LCM). These techniques can be applied to studying miRNA in other species, tissues, and cell types. Includes: Obtain Laser Capture Microdissected Samples; Isolate miRNA from LCM Samples; Quantitate miRNA by qRT-PCR. - [Read Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples]

Category: PCR Protocols: PCR Optimization

Protocol for enhancing PCR of very difficult regions. - [Read Enhancing PCR of Very Difficult Regions Protocol]

Category: Microarray Protocols: Microarray Analysis Protocols

Genome-wide analysis of data generated on the Affymetrix 10K Xba 142 arrays for identification of regions with high probability to contain genes responsible for Micronodular (non-pigmented) Adrenocortical Hyperplasia. - [Read Genome-Wide Analysis Protocol]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Chromatin-IP (or ChIP) has been a useful method for the localization of proteins to specific regions of DNA. ChIP applied to microarrays (ChIP on Chip) Brown Lab - [Read Immunoprecipitation of Chromatin from Fixed Yeast Protocol PDF]

Category: Nucleic Acid Purification Protocols

Preparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded DNA probe.  At the end of the reaction nuclease S1 is used to degrade unhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by autoradiography or Southern hybridization.  Method used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates. - [Read Mapping RNA with Nuclease S1 Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol describes a method for quantitative measurement of DNA using propidium iodide (PI) staining and flow cytometry. PI stains all double-stranded regions of both DNA and RNA by intercalating between the stacked bases of the double helix. PI cannot penetrate an intact cell membrane; therefore, cells are fixed prior to staining. The ethanol-fixed cells can be stored unstained at 4°C for days, or even weeks, and then stained and analyzed. - [Read Measurement of DNA Content Using Propidium Iodide (PI) Staining of Fixed Whole Cells Protocol]

Category: Molecular Imaging

NIH Image. Image can be used to measure area, mean, centroid, perimeter, etc. of user defined regions of interest. It also performs automated particle analysis and provides tools for measuring path lengths and angles. Spatial calibration is supported to p - [Read NIH Image for Mac]

Category: PCR Protocols: PCR Optimization

Procedure details the establishment of an amplification procedure for GC-rich sequences.  The DNA fragments of interest are amplified in the presence of either 5% DMSO, 1 M betaine, 2 M betaine, 1 M betaine, and 5% DMSO; 2 M betaine and 5% DMSO; 0.4 M tetramethylene sulfone; or without any of the enhancers. - [Read PCR Amplification of Highly GC-Rich Regions Protocol]

Category: Protein Protocols: Protein Extraction From Tissue

Combination of nucleic acid and protein isolation with tissue array construction: Using defined histologic regions in single frozen tissue blocks for multiple research purposes - [Read Protein isolation with tissue array construction]

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

The first part of the isolation procedure is a flotation through a continuous iodixanol gradient; this gradient is essentially a resolving gradient in which the caveolin-rich vesicles are concentrated in the top third of the gradient, while the predominantly caveolin-poor vesicles band in denser regions. A second discontinuous gradient is essentially a concentration gradient to band the caveolin-rich vesicles sharply at an interface. - [Read Purification of Caveolae Membranes from a Plasma Membrane Fraction of Cultured Cells and Tissues]

Category: DNA Protocols: DNA Sequencing Protocols

Protocol for sequencing difficult regions on DNA. - [Read Sequencing Difficult DNA Regions Protocol]

Category: Cytogenetics Protocols

Silver Staining for Nucleolar Organizer Regions. Trude Schwarzacher and Molecular Cytogenetics Research Group - [Read Silver Staining for Nucleolar Organizer Regions]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes a method to stain nerve fibers in tissue slices of avian embryos using an antibody against the 160-kD subunit of neurofilaments.  This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos.  The tissue is cut in slices using a vibratome or tissue slicer.  The protocol is suitable for older embryos after approximately stage 33 and regions that are not accessible by whole-mount analysis. - [Read Staining of Tissue Slices for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos]

Category: Avian Bird Protocols

Protocol describes a method to stain nerve fibers in tissue slices of avian embryos using an antibody against the 160-kD subunit of neurofilaments. This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos. The tissue is cut in slices using a vibratome or tissue slicer. The protocol is suitable for older embryos after approximately stage 33 and regions that are not accessible by whole-mount analysis. - [Read Staining of Tissue Slices for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos Protocol]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Immunoprecipitation Protocols

Protocol describes a method for the detection of proteins bound to specific regions of chromatin in yeast. There are many variations of this assay. - [Read Yeast Chromatin Immunoprecipitation (ChIP) Assay Protocol]