designed Protocols

Category: Cell Culture Protocols: Insect Cell Culture Protocols

This protocol is designed to adapt High Five™ (BTI-Tn-4) cells to serum-free suspension culture in HyQ SFX-Insect in 5 passages. The entire adaptation process takes approximately two weeks and the resulting adapted cells have doubling times of 16 and 20 hours and minimal clumping in suspension culture. - [Read Adaptation of High Five™ Cells to HyQ© SFX Insect Medium]

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

There are many ways to adapt cell lines to serum-free media. Five methods are presented that are designed for adapting hybridomas to a protein-free medium. These protocols may require some modifications for your particular cell line and conditions. - [Read Adapting Cells to a Serum-Free Environment Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

AFLP was designed as a highly sensitive method for DNA fingerprinting to be used in a variety of fields. We are using this technology to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been identified genetically by mutant phenotype. Protocol includes: Generate polymorphic recombinant F2 (or F3) population; Isolate genomic DNA; Restriction of DNA; Ligation of adapters; Pre-amplification of template DNA; AFLP-PCR; etc. - [Read AFLP For Positional Cloning]

Category: PCR Protocols: cDNA Synthesis Protocols

An oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR.  Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs - [Read Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol]

Category: Cell Biology and Cell Culture

Protocol applies EFs to cells in vitro but has been modified and to use electrotactic chambers to accommodate cells growing in planar culture or in three-dimensional (3D) gels, en bloc tissue cultures in 3D and possible small embryos, such as that from frog and zebra fish. The EF is applied to the cells or tissues cultured in a customer designed electrotactic chamber via agar salt bridges, Steinberg’s solution and Ag/AgCl electrodes. - [Read Application of Direct Current Electric Fields to Cells and Tissues in vitro]

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: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor.  This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest.  Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out. - [Read Competitive RT-PCR: Preparation of Competitor RNA Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Specimen chambers have had many designs published over the years describing systems that offer excellent optical properties while allowing specimens to be maintained for varying amounts of time. Ranging in complexity from the simple preparation of a sealed coverslip on a microscope slide to sophisticated perfusion chambers that enable tight control of virtually all environmental variables culture chambers are designed to to allow living specimens to be observed with minimal invasion at high res. - [Read Culture Chambers for Live-Cell Imaging]

Category: Fluorescent Dyes Protocols

Cyanine dye reagents are useful as fluorescent labels for proteins. This protocol has been designed to label the thiol group on cysteine using Cy3 or Cy5 minimal maleimide labeling dyes. - [Read Cyanine Dye (Maleimide) Protein Labeling Protocol]

Category: siRNA and RNAi Protocols

dsRNA From Clones, Primer Designed dsRNA. Drosophila RNAi Screening Center at Harvard Medical School - [Read dsRNA Synthesis Protocol]

Category: Cell Biology and Cell Culture

Cell fractionation of cellular components using Percoll a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. Percoll becomes a simple matter to establish a linear density gradient. Organelle separations are much easier to accomplish on Percoll density gradients than on sucrose gradients. - [Read Equilibrium Density Gradient Percoll Protocol]

Category: Molecular Model Organisms: Mouse Animal Protocols: Mouse Genotyping Protocols

24-mer A=4, C=10, G=2, T=8, HLA-A2.1 ( 003475) primer designed in Mouse Genotyping lab. ... This genotyping protocol is used for the following strains: ... - [Read Genotyping Protocol for TgN(HLA-A2.1)1Enge]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

This test is designed to detect irreversible toxic effects on both cell growth and survival, by the evaluation of colony-forming (CF) efficiency, in hepatoma cell lines derived from man, rat and mouse. - [Read Hepatoma Cell Cultures as In Vitro Models for Hepatotoxicity]

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

Protocol for in vitro transcription and translation using the coupled reticulocyte lysate system. This protocol is designed to test random samples on a protein gel. Scale up the reactions accordingly. Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read In Vitro Transcription and Translation Using the Coupled Reticulocyte Lysate System]

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: Cell Biology and Cell Culture: Cell Culture Growth Media: Mammalian Cell Culture Growth Media Protocols

The culture medium is an essential component of the in vitro environment and must be selected or designed with care. This protocol provides guidelines for design of serum-containing and serum-free media, selective and specialty media, and media for growth under special conditions such as soft-agar growth. - [Read Media for Culture of Mammalian Cells Protocol]

Category: DNA Microarray Protocols

The "7 keys to successful microarray analysis" has been designed to guide researchers step-by-step through the design, implementation, analysis, and publication of a microarray experiment. - [Read Microarray Success]

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

Protocol describes a system which includes all of the necessary components for in vitro transcription as well as a positive control template that provides run-off transcripts from a CMV immediate early promoter. This system is designed for runoff transcription. Alternatively, transcription products can be analyzed by primer extension. - [Read Nuclear Extract in vitro Transcription System]

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: Fungi Protocols

Protocol for the preparation of solid tissue for Aspergillus galactomannan antigen detection by Platelia (Biorad). Technique was designed for use on human serum. However, it may also be possible to perform this method on solid tissues and organic solutions. Viscous solution and tissue specimens need to be pre-treated to achieve the extraction of the Aspergillus antigen and to get a homogeneous sample in solution. - [Read Preparation of Solid Tissue for Aspergillus Galactomannan Antigen Detection by Platelia Protocol]

Category: C. elegans Protocols

Protocol was designed to rapidly generate small scale cytosolic extracts of C. elegans for Western or IP (has not been tested for RNA work). The protocol works well for between 50 to 5000 worms and has not been extensively tested on larger a scale, though it should work. Includes: Collection; Sonication; Clearing lysate; Immunoprecipitation. - [Read Preparation of Worm Extracts by Sonication Protocol]

Category: Fungi Protocols: Neurospora Protocols

Procedure for preparing and transforming spheroplasts of Neurospora crassa. The procedure is designed to produce a large number of spheroplasts to be stored frozen at -80°. - [Read Preparing and Transforming Spheroplasts of Neurospora crassa Protocol]

Category: Fungi Protocols

Protocol for bird medium. This medium was designed to circumvent some problems that arise in the use of Medium N. - [Read Protocol for Bird Medium: Alternative to Vogel Medium]

Category: Cell Biology and Cell Culture: Endothelial Cell Protocols

Primary mammalian endothelial cells protocol. This protocol is designed for primary endothelial cells isolated from various organs of mammals. Large and flat cells, often with large nuclei. Includes: Required reagents; DNA preparation and quality; Preparation of cells and cell culture; Important controls; Nucleofection protocol. - [Read Protocol Primary Mammalian Endothelial Cells]

Category: Transgenic Mouse Protocols: Mouse Genotyping Protocols

PCR screens must be designed to detect transgene DNA at the single copy level.Copy standards are prepared by mixing non-transgenic tail DNA with a known amount of transgene DNA to produce transgene copy standards. University of Michigan Transgenic Animal - [Read reparation of Copy Standards for Southern Blot Copy Number Determination]