products Protocols

Category: Cell Culture Protocols: Cell Immortalization Protocols

Cultivating animal cells in the laboratory is an indispensable technique for cell biologists. However, most normal primary cell lines, while faithfully reproducing the phenotype of their tissue of origin, do not grow indefinitely in culture. After a series of population doublings (the number of which varies by species, cell type, and culture conditions) primary cells enter a state where they no longer divide. - [Read Immortalization of Cells in Culture]

Category: Protein Protocols: Western Blot Protocol

Nice Western Blotting Protocol using some eBioscience Products. - [Read Immunoblotting Using eBioscience Products]

Category: RNA Protocols: RNA-binding Protein Purification

MACS Streptavidin Kit for purifying RNA-binding factors. See pdf below. Miltenyi Biotec - [Read MACS Streptavidin Kit]

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Dnase I is used to fragment a radiolabeled target DNA in the presence and absence of a nuclear extract.  A "footprint" is generated when a protein binds to the target and protects a specific segment of DNA from the nucleolytic activity of Dnase I. By comparing the electrophoretic mobility of the Dnase I cleavage products to those of a sequence ladder derived from the same DNA fragment, the position(s) of the DNA sequences recognized by DNA-binding proteins can be determined. - [Read Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol]

Category: Reporter Gene Assays: Cat Assays Protocols

In this protocol, extracts prepared from cells transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid are incubated with radiolabeled chloramphenicol. The acetylated products generated by the action of CAT are separated from the unmodified drug by thin-layer chromatography and quantitated by scraping the spots from the thin-layer plates and counting them by scintillation spectroscopy. - [Read Measurement of CAT in Extracts of Mammalian Cells Using Thin-layer Chromatography]

Category: DNA Microarray Protocols

This protocol describes PCR amplification of eukaryotic cDNA plasmid inserts, gel electrophoresis, purification, and storage of PCR products. Hasseman. TIGR Microarray Protocols. - [Read MICROARRAY PCR, PURIFICATION, AND STORAGE]

Category: PCR Protocols: PCR Cloning Protocols

The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments.  This protocol describes the strategies for generating and manipulating suitable ends on the PCR fragments. - [Read Molecular Cloning of PCR Products Protocol]

Category: DNA Protocols: DNA Sequencing Protocols: Big Dye Protocols

Protocol describes the purification, quantification, and subsequent sequencing of amplified DNA fragments using PCR. Excess nucleotides are removed from the initial PCR products using spun columns, and the products are quantified using fluorometry. - [Read Nonradioactive Cycle Sequencing of PCR-Amplified DNA Protocol]

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: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

One step extraction for isolation of plant DNA. DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm2 in less than 20 min & no tube changes. Method was tested on several plant species. Method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated using standard DNA isolation. - [Read One-Step Isolation of Plant DNA Suitable for PCR Amplification]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: PCR Optimization Protocols

The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. P - [Read PCR Program Design]

Category: PCR Protocols: PCR Optimization

The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. P - [Read PCR Program Design]

Category: Molecular Biology Protocols: Carbohydrate Protocols

Pierce Protein Glycosylation Kits and Protocols. Pierce - [Read Pierce Protein Glycosylation Kits]

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol describes a method for introducing gene constructs into mouse embryos by electroporation. Gene constructs can be quickly tested for tissue-specific transcriptional activity or can be used to overexpress gene products. - [Read Protocol Electroporation]

Category: RNAi Technology Protocols

Short interfering RNAs (siRNAs) can be used to prime RNA synthesis by the RNA-dependent RNA polymerase (RdRP).  SiRNAs can be used by RdRP as primers for specific cellular mRNAs, forming dsRNA products capable of inducing transitive RNAi. - [Read Protocol for siRNA-Primed RNA Synthesis Protocol]

Category: Cloning Protocols

Protocol describes how to use proteinase K to destroy thermostable enzymes and to purify amplified DNA in preparation for cloning. - [Read Purification of PCR Products in Preparation for Cloning Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols

This protocol describes how to use proteinase K to destroy thermostable enzymes and to purify amplified DNA in preparation for cloning. - [Read Purification of PCR Products in Preparation for Cloning Protocol]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

Quality is important in all aspects of tissue culture since the quality of materials used i.e. media and other reagents) will affect the quality of the cultures and products derived from them. The main areas of quality control that are of concern for tissue culture are: The quality of the reagents and materials; The provenance and integrity of the cell lines; The avoidance of microbial contamination. - [Read Quality Control Considerations for Cell Culture]

Category: RNA Protocols: cDNA Libraries Library

Hoskins et al. 2005 NAR. "... describe a PCR-based method for directed screening of plasmid cDNA libraries. " - [Read Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP)]

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

An in vitro red blood cell assay is presented which allows the estimation of the irritation potential of tensides and tenside containing materials such as shampoos, shower gels, cleaning products, etc. The estimation is based on the fact that surfactants interact strongly with cellular membranes and proteins.  Both effects are measured photometrically by use of the inherent native dye, oxyhemoglobin. - [Read Red Blood Cell Test System Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In the first part of this protocol, the linear range of amplification is determined by carrying out 10 identical PCRs in the presence of [{alpha}-32P]dCTP and stopping one reaction after every two cycles.  Amplification products are quantified on a denaturing polyacrylamide gel and the results plotted on a graph (counts per minute vs. cycle number).  Total RNA is used as an internal control. - [Read Relative RT-PCR: Determining the Linear Range of Amplification and Optimizing the Primers:Competimer]

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

In vitro transcription systems includes instructions for use of products P1420, P1430, P1440, P1450 and P1460.Includes information and protocols on RNA Transcription in vitro. Information on DNA Template Preparation;Synthesis of High-Specific-Activity Radio labeled RNA Probes;Determining Percent Incorporation and Probe Specific Activity;Removal of the DNA Template Following Transcription;Removal of unincorporated nucleotides;Synthesis of large amounts of RNA;Capping RNA for in vitro translation. - [Read Riboprobe In Vitro Transcription Systems]

Category: siRNA and RNAi Protocols

siRNA Transfection Protocol for TransPass R1 NEB - [Read siRNA Transfection Protocol for TransPass R1 NEB]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

A vector that lacks inserts can significantly contaminate a DNA library. This contamination can occur either from self-ligation of single-cut vector or from uncut circular starting plasmid. - [Read Synthesis of DNA Libraries: Use of PCR to Analyze Ligation Substrates and Products]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Technique yields a heterogeneous population of short radiolabeled molecules 200-300 nucleotides in length. These probes are synthesized, as in Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates, by extension of an oligonucleotide primer on a single-stranded DNA template. The radiolabeled products of the reaction are then separated from the template by electrophoresis through a denaturing gel from which they are eluted directly into hybridization buffer. - [Read Synthesis of Single-stranded DNA Probes of Heterogeneous Length from Bacteriophage M13 Templates]