system Protocols

Category: Molecular Model Organisms: Yeast Two-Hybrid Screen Protocol

Yeast Two-Hybrid System. TRAFO Solutions. Gietz Lab - [Read 2 Hybrid System TRAFO Protocol]

Category: PCR Protocols

The MagneSil system can selectively isolate PCR products that are more than 150-bp long from primers and primer -dimers.  The technology can be used with a number of robotic workstations, including Beckman Coulter’s Biomek 2000 and FX Laboratory Automation Workstations.  The procedure can also be carried out manually.  Typical recovery is more than 80% for a 1-kb product with negligible carryover of primers or nucleotides. - [Read A Magnetic Particle-Based Method for Purifying PCR Products from Solution Protocol]

Category: DNA Protocols: EMSA DNA-Protein Protocols

A non-radioactive electrophoretic mobility shift assay based on the digoxigenin detection system had been developed and applied to the analysis of the interaction between the heat shock transcription factor and the heat shock element of N. crassa. (Bioch - [Read A non-radioactive electrophoretic mobility shift assay for the detection of heat shock element (HSE)]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol describes typical methods that are used to propagate and purify AAV vectors for experiments both in vitro and in vivo. Includes: Principles of the Triple Plasmid Transfection System; Plasmids; Transfection and Extraction of Virus; Purification of the AAV vector. - [Read A Protocol for AAV Vector Production and Purification]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

A simplified system for rapid generation of recombinant adenoviruses. Includes: Generation of Recombinants in Bacterial Cells; Viral Production in 293 or 911 Cells; Preparation of High Titer Viral Stocks. - [Read A Simplified System for Rapid Generation of Recombinant Adenoviruses]

Category: Cloning Protocols: Vector Protocols

Protocol is used to propagate and purify AAV vectors for experiments both in vitro and in vivo. Includes: Principles of the Triple Plasmid Transfection System; Plasmids; Transfection and Extraction of Virus; Purification of the AAV vector. - [Read AAV Vector Production and Purification Protocol]

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

Protocol used to for immunohistochemistry on paraffin-embedded sections. Based on use of microwave energy to effect antigen retrieval. The immunohistochemistry procedure, is for use of Biomeda's HistoScan kit based on a streptavidin-peroxidase/biotinylated second antibody detection system with 3-amino, 9-ethylcarbazole (AEC) as chromogen. Undoubtedly, other kits or home-made reagents will also work . - [Read Antigen Retrieval for Immunohistochemistry with Paraffin-Embedded Tissues Protocol]

Category: Immunology: B Cell Protocols

Describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. - [Read Assays for B Lymphocyte Function Protocols]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

Choosing a cell viability or cytotoxicity assay from among the many different options available can be a challenging task. Includes information on: Establishing an In Vitro Model System; Choosing an Endpoint to Measure; Characterizing Assay Responsiveness; Determining Dose and Duration of Exposure; Homogeneous Assays for Multiwell Formats and Automated Screening; Additional Factors to Consider When Choosing a Cell Viability Assay; Cell Viability Assays that Measure ATP Protocol; etc.. - [Read Cell Viability Information For Protocols and Applications]

Category: Immunology

Protocol for detection of autoantibodies with self-assembling radiolabeled antigen tetramers. Details how to produce radiolabeled antigen-streptavidin tetramers for detection of antibodies by immunoprecipitation. Optionally, the antigen tetramers can be denatured to compare responses to folded and unfolded antigen in the same system. This technique can be applied to a large or small number of samples, and a given sample can be simultaneously assayed with multiple antigens. - [Read Detection of Autoantibodies with Self-Assembling Radiolabeled Antigen Tetramers Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probes. - [Read Detection of mRNA on Paraffin Embedded Material of the Central Nervous System with DIG-Labeled RNA]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNAs on cryosections of the cardiovascular system using DIG-labeled RNA probes. Protocol was optimized from a protocol using 35S-labeled RNA probes. It allows to detect the expression of low abundant mRNAs in the cardiovascular system, e.g. of the proinflammatory cytokine GM-CSF in normal human coronary arteries, and of IL6 and gp130 in human failing hearts. The protocol can be combined with immunohistochemistry. - [Read Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol describes here a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. Includes: In situ hybridization with DIG-labeled probes; Detection of DIG-labeled probes; Fluorescence microscopy. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol describes a high sensitivity indirect detection procedure for DIG-labeled hybridization probes.  The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization.  This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes. 
    - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol for dual luciferase reporter system. Includes: Transfections; Cell Lysis; Reagent Preparation; Luminescence measurement; Wash injectors. - [Read Dual Luciferase Reporter System Protocol (PROMEGA)]

Category: Luciferase and Dual Luciferase Assays

Dual Luciferase Assay System. Promega. PDF - [Read Dual Luciferase Assay System. Promega. PDF]

Category: Molecular Model Organisms: Yeast Two-Hybrid Screen Protocol

Interactor Hunt Protocol. Testing whether a bait activates transcription. Screening a library for interactors. Finley Lab. 1997. - [Read EXAMINING THE FUNCTION OF PROTEINS AND PROTEIN NETWORKS WITH THE YEAST TWO-HYBRID SYSTEM]

Category: Bacterial Cell Culture Protocols: Bacterial Cell Culture Media Protocols

This protocol has been used successfully to 15N or 13C/15N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts: - [Read Expression Protocol in M9 Minimal Media via T7 Promoter]

Category: PCR Protocols: cDNA Synthesis Protocols

This cDNA synthesis system simplifies your work dramatically.  All reaction components are premixed and lyophylised.  You have to add your RNA and (for Your-Prime beads) the primer.  Another advantage of the system is a little number of pipetting steps required, and therefore reduced risk of Rnase contamination and RNA degradation. - [Read First strand cDNA synthesis with Ready-To-Go Beads Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension. Hybridization technique which does not need formamide and dextran sulfate. As a model system, we used the repetitive specific human DNA probe pUC 1.77, labeled it with digoxigenin-11-dUTP by nick-translation, and hybridized it to metaphase chromosomes in suspension. These chromosomes were isolated by standard techniques from human lymphocytes. - [Read Fluorescence In Situ Hybridization of a Repetitive DNA Probe to Human Chromosomes in Suspension]

Category: Chromatography Protocols

FPLC Protocol. The FPLC consists of a pump and a column which will withstand high pressure so separations can be carried out relatively quickly. For a detailed description there is a FPLC system handbook which is particularly useful for trouble shooting. For use of individual columns follow the "instructions" (in the green folder) which accompany each one. Sir William Dunn School of Pathology, Oxford University. - [Read FPLC Protocol]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for constant-flow microinjection using the Pneumatic PicoPump (World Precision Instruments). This type of system is very simple and can be assembled on a relatively low budget. In this method, a constant flow of sample is delivered from the tip of the pipette, and the amount of sample injected into the cell is determined by how long the pipette remains in the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Controlled-Flow System Protocol]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for pulsed-flow microinjection using the Eppendorf FemtoJet injector and Eppendorf InjectMan; this is the most common type of pulsed-flow microinjection system currently being used. The advantage of this type of system over a controlled-flow system is that much more control is available over the injection parameters, reducing variability in injections. In addition, the system allows a diagonal insertion of the needle into the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System Protocol]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for the generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. - [Read Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

Accumulation of lipophilic substances in the plasma membrane may affect the membrane lipid order and consequently affect the function of these proteins.  Changes in the activity of the Na+/K+ -ATPase, which is the major active transport system responsible for the electrochemical potential in mammalian cells, can therefore be an indication of the effect that a chemical may have on the viability of the cell membrane and possibly the whole cell. - [Read Hamster Ovary Cell NA+/K+ -ATPase Test]