preparation Protocols

Category: Yeast Protocols

Protocol for in vitro endoplasmic reticulum to golgi transport reaction in Yeast. Includes: Preparation of Membranes; One-Stage Reaction; Two-Stage Reaction using Normal Amount of Membranes; Two-Stage Reaction using Low Concentration of Membranes. - [Read In Vitro Endoplasmic Reticulum to Golgi Transport Reaction in Yeast Protocol]

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Protocol for in vitro footprinting methods. Includes: Preparation of labeled probes; Gel purification. - [Read In Vitro Footprinting Methods Protocol]

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

With this protocol, transcripts that were initiated from specific genes by RNA polymerases prior to permeabilization can be measured. Instead of a nuclear extract, permeabilized cells are used. Includes information on: Permeabilization of Cells; In vitro Transcription Reaction (Run-off); Isolation of RNA; Preparation of Slot Blot Membrane for Hybridization; Hybridization of Nitrocellulose Membrane; TCA Precipitation to Determine Incorporation of [32P] GTP into Nucleic Acid - [Read In Vitro Transcription Assay (Run-off Assay) using Permeabilized Cells]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

Protocol by using the technique of Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca2+. Protocol includes: Preparation of Indo-1 and preparation of Ionomycin. - [Read Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca2+]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Interphase nuclei preparation for FISH. Sanger Institute. - [Read Interphase nuclei]

Category: PCR Protocols: Inverse PCR Protocols

Inverse PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available.  The technique involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region.  The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in the PCR. - [Read Inverse PCR Protocol II]

Category: Plant Biology Protocols: Arabidopsis Nucleic Acid Protocols

Includes: Isolation of Arabidopsis LEU2 cDNAs by complementation of the yeast leu2 mutation; Recovery of plasmid DNA from yeast cells; Analysis of Arabidopsis cDNAs that complement a yeast leu2 mutation; Preparation of library DNA from the Lacroute cDNA library; Preparation of Yeast Media. - [Read Isolation of Arabidopsis cDNAs by Complementation in Yeast]

Category: BACs Bacterial Artificial Chromosome Protocols

BAC DNAs are prepared from 5-ml cultures of BAC-transformed cells by a modification of the standard alkaline lysis method (Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation). The yield typically varies between 0.1 and 0.4 µg of BAC DNA. - [Read Isolation of BAC DNA from Small-scale Cultures Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination. - [Read Isolation of Plant Transcription Factors Using a Modified Yeast One-Hybrid System]

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

Protocol describes the purification release of plasmid DNA from yeast cells. The resulting plasmid DNA can be used to transform Escherichia coli or yeast. - [Read Isolation of Plasmid DNA from Yeast Cells: A Ten-Minute Preparation]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes a plate method that gives very good yield for cloning. Includes: T-TYN Media + Mg+2; T-TYN Plates; T-TYN Top Agarose. - [Read Lambda DNA Preparation Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols

Protocol for lambda DNA preparation. This is a plate method that gives very good yield for cloning. - [Read Lambda DNA Preparation Protocol]

Category: Protein Protocols: Nuclear Extraction Protocols

Homogenize cells or tissue, Pellet nuclei, Lyse nuclei, Reprecipitate nuclear proteins, dialyze nuclear extract. Hattori M et al., DNA Cell Biol. 1990. Homogenization buffer and dialysis buffer recipes. - [Read Large scale nuclear extract preparation]

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol describes the use of agarose plugs for isolation of yeast artificial chromosome (YAC) DNA. The DNA can then be run on a pulsed-field gel and used for microinjection to produce transgenic mice. - [Read Large-Scale Preparation of Agarose Plugs of Yeast DNA Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol used chiefly to generate large stocks of double-stranded DNA of strains of M13 that are routinely used as cloning vectors. Large amounts of single-stranded DNA of an individual recombinant may occasionally be needed for specific purposes, e.g., to generate many preparations of a particular radiolabeled probe or to construct large numbers of site-directed mutants. - [Read Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol]

Category: Yeast Protocols: Yeast Transformation Protocols

Protocol for large-scale yeast transformation. Includes: Yeast Cell Preparation; Large Scale Transformation; To plate on solid medium; To select in liquid. - [Read Large-Scale Yeast Transformation Protocol]

Category: Histology Protocols: Laser Capture and Microdissection

Preparation of Frozen Tissue for LCM, Preparing Sections from Frozen Tissue for Laser Microdissection, Preparing Sections from Fixed, Paraffin-embedded Tissue for Laser Microdissection Protocols and methods. Laser Microdissection Laboratory. UAB. - [Read Laser Capture Microdissection Protocols]

Category: Buffers Media, and Solutions: Bacterial Solutions

LB Agar Preparation. Sosnick Group. - [Read LB Agar Preparation]

Category: Microbiology: Bacterial E.Coli Culture Media & Plates

LB Plates Pouring and Preparation. antibiotics addition. Janet Smith, Boston Biomedical Research Institute - [Read LB Plates Pouring and Preparation]

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

Lipoplex (cationic liposome-DNA complex) is formed via electrostatic interaction of anionic nucleic acids with cationic liposomes. A thin film of lipids is dried on the bottom of a glass tube and rehydrated in an aqueous solution. The resulting liposome suspension is passed through polycarbonate filters of desired pore size. This protocol also describes the preparation, physical properties, and biological activity of liposome-polycation-DNA (LPD) nanoparticles. - [Read Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery Protocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Caenorhabditis elegans, a small (adults are ~1 mm long), free-living soil nematode that feeds on bacteria, is an ideal organism for applying various live microscopy techniques. This protocol describes useful techniques for preparing C. elegans for live microscopic analysis. Details of sample preparation depend on the developmental stage of the worm to be studied. - [Read Live Imaging of Caenorhabditis elegans: Preparation of Samples]

Category: C. elegans Protocols

Protocol describes useful techniques for preparing C. elegans for live microscopic analysis. Details of sample preparation depend on the developmental stage of the worm to be studied. - [Read Live Imaging of Caenorhabditis elegans: Preparation of Samples]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

Assay measures cell viability. It is a two-color fluorescence assay that simultaneously determines Live cell number and Dead cell number. This protocol is designed for use with the GEMINI XS Microplate Spectrofluorometer, a multi-well plate scanner with dual excitation/emission capabilities, but the assay is also adaptable for flow cytometry and fluorescence microscopy. Includes: Cell Culture; Preparation for the Assay; Live/Dead Assay; Reading the Plate; Data Analysis; Alternative protocol. - [Read Live/Dead Assay for Cell Viability Protoco]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Luciferase assay using a 24 well plate. Includes: Cell Lysis; Reagent Preparation; Luminescence measurement; Wash injector; Protein quantitation; Data Analysis. - [Read Luciferase Assay 24 Well Plate (Promega System)]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the first step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LC-MS/MS.  This procedure is used to prepare protein extracts from WEHI-231 cells.  This preparation method provides total cellular protein samples that are free of contaminating nucleic acids. - [Read Lysis and Protein Extraction from WEHI-231 Cells with TriPure Isolation Reagent Protocol]