interest Protocols

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: Drosophila Protocols: Drosophila Genetics Protocols

Protocol describes how to test whether a transcription factor disrupts the chromatin of a promoter of a gene of interest. First, chromatin is assembled in vitro on the gene of interest in the presence and absence of a transcriptional activator (see Protocol on Assembly of Chromatin with Drosophila S-190 Chromatin Assembly Extract and Transcriptional Activators). - [Read Nucleosomal Array Disruption Assay Protocol]

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: PCR Protocols

DNA prepared by PCR-mediated gene disruption can be used to transform yeast in gene replacement experiments.  This protocol uses two primers, tailed with approximately 50 nucleotides homologous to the gene of interest, that target insertion of the PCR product to that locus.  Each primer ends with a universal sequence that is designed to amplify various selectable markers from plasmid templates. - [Read PCR-Mediated Gene Disruption: One-Step Method Protocol]

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: Viral Culture and Isolation Protocols: Viral Culture Protocols

In this protocol, a bacterial lysogen is constructed from a recombinant bacteriophage {lambda} encoding a fusion protein of interest. The resulting lysogenic colonies are induced to synthesize the fusion protein, which is then isolated in preparation for functional and biochemical analyses. - [Read Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda} Lysogens]

Category: Laboratory Model Organisms Protocols

Genetic Protocols for Pristionchus pacificus. Includes: Freezing worms; EMS mutagenesis; Psoralen mutagenesis; Construction of deletion libraries to generate P. pacificus gene knock-outs; Designing primers for the gene of interest; RNAi and morpholino by injection. - [Read Pristionchus pacificus Genetic Protocols]

Category: Reporter Gene Assays: GFP Assay Protocols

In preparation for FLIM-FRET analysis, the appropriate donor and acceptor components must be introduced into live or fixed cells. The method of introduction depends on the nature of the components and the state of the cells. For example, plasmid DNAs encoding a protein of interest fused to a variant of GFP may be introduced into live cells by transfection or microinjection, whereas labeled antibodies are delivered by microinjection. - [Read Probing Protein Interactions Using GFP and FRET Protocol]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Perform this protocol after determining the imidazole concentration that yields the best purification of the recombinant protein of interest. - [Read Protein Purification Using Immobilized Metal-Ion Affinity Chromatography under Optimized Imidazole]

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

In this protocol nuclei isolated from cells expressing the gene of interest are incubated with radiolabeled UTP which is incorporated into nascent RNA transcripts by RNA polymerase molecules that were actively transcribing at the time the cells were harvested. Because very little denovo initiation of RNA synthesis occurs in isolated nuclei, transcription of the target gene can be measured by hybridizing the radiolabeled RNA to an excess of the target gene immobilized on a nitrocellulose or nylon - [Read Protocol for Transcriptional Run- On Assays]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol describes a general method for the purification of protein using dye-ligand affinity chromatography.  The effectiveness of this protocol is dependent upon the reader having previously determined the optimal dye and buffer conditions to use for binding and eluting the protein(s) of interest. - [Read Purification of Protein Using Dye-Ligand Affinity Chromatography Protocol]

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes the use of RT-PCR to confirm knockdown of a gene of interest in RNAi-treated mouse oocytes and early embryos.  All of the solutions used during RNA isolation and reverse transcription must be Rnase-free - [Read RNA Isolation and RT-PCR from dsRNA-Treated Mouse Oocytes and Early Embryos Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

A powerful way to identify a mutation in the gene of interest and to test mutant plants for phenotypes that are predicted to result from loss of function of that gene is by PCR screening. Pools of insertion lines are screened using one primer corresponding to the gene of interest and one primer corresponding to the end of the insertion element. The synthesis of a product indicates the presence of an insertion in the gene of interest. - [Read Screening DNA Pools for T-DNA Insertions in Arabidopsis Genes Protocol]

Category: DNA Protein Interactions Protocols

The multiprotein-DNA complex of interest is formed using the site-specifically derivatized DNA fragment.  The complex is then UV-irradiated, initiating covalent cross-linking with proteins in direct physical proximity to the cross-linking agent.  Extensive nuclease digestion is performed to eliminate uncross-linked DNA and convert cross-linked DNA to a cross-linked, radiolabeled nucleotide "tag." - [Read Site-Specific Protein-DNA Photo-Cross-Linking: Analysis of Structural Organization of Protein-DNA]

Category: Cloning Protocols: Vector Protocols

Protocol for the transfection/Retroviral Infection pCL-Eco+Gene of interest into 293T cells infecting HC11 cells. Includes: Split 293T cells for transfection; Transfect 293T cells; Split HC11 cells for infection; Infect target (HC11) cells. - [Read Transfection/Retroviral Infection pCL-Eco+Gene of Interest into 293T Cells Infecting HC11 Cells]

Category: Viral Manipulation Protocols: Phage Protocols

Protocol is used to identify and isolate recombinants containing DNA sequences of interest. - [Read Transfer of Bacteriophage DNA from Plaques to Filters Protocol]