vitro Protocols

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: 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: Transfection Protocols

Protocol describes transfection of plasmid DNA into mammalian cell lines using electroporation, a process whereby external application of electric pulses induce cell membrane permeability. Cells in suspension and small volume cells are difficult to transfect, whereas adherent cells and large volume cells are relatively easy. Regardless of cell size or phenotype, transfection efficiency increases with a high concentration of cells in a small volume. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol describes transfection of plasmid DNA into mammalian cell lines using electroporation, a process whereby external application of electric pulses induce cell membrane permeability. A number of factors can affect electrotransfection efficiency. In general, cells in suspension and small volume cells are difficult to transfect, whereas adherent cells and large volume cells are relatively easy. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Peroxisome Isolation Protocols

To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. - [Read Peroxisomes in Saccharomyces cerevisiae]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes a method to prepare and fractionate Drosophila embryo extracts.  These extracts can be used for the in vitro characterization of RNAi. - [Read Preparation and Fractionation of Drosophila Embryo Extracts for the In Vitro Characterization of RNA]

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

Protocol describes in vitro transcription with the SP6 polymerase.  For other polymerases (T3, T7), simply use the appropriate buffer and desired RNA polymerase instead of the SP6 polymerase. - [Read Preparation of dsRNA Molecules for RNAi in Mouse Oocytes and Early Embryos Protocol]

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

Protocol for preparation of KC nuclear extract for in vitro splicing. Protocol makes 3.4 ml of extract for every 4 liter of cells (depending on initial cell concentration). Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read Preparation of KC Nuclear Extract for In Vitro Splicing]

Category: Cytoskeleton Protocols: Microtubules Protocols

Segmented and polarity-marked microtubules are very useful for many different types of in vitro assays. Segmented microtubules are microtubules with a bright seed and dim elongated segments on both ends. Polarity marked microtubules are microtubules with a bright seed and a dim elongated segment only on one end -- the plus end. - [Read Preparation of Segmented and Polarity Marked Microtubules Protocol]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

siRNAs produced upon the addition of dsRNA to Drosophila embryo extract are enriched in a micrococcus-nuclease-resistant fraction.  After proteinase K treatment and dephosphorylation with calf intestinal phosphatase, these siRNAs mediate efficient RNAi in vitro. - [Read Preparation of siRNAs from Drosophila Embryo Extracts Protocol]

Category: Developmental Biology: Embryology Protocols

Procedures for in vitro production of bovine embryos. Includes: Collection of Ovaries; Oocyte Collection; Preparation of IVF Medium; In Vitro Fertilization; Culture of Embryos. - [Read Procedures for In Vitro Production of Bovine Embryos]

Category: Avian Bird Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes. The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII). The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. After purification, the RNA strands are annealed to yield a dsRNA molecule suitable for RNAi in avian embryos. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes.  The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII).  The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

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

Protocol includes information on: Preparation of Membranes; One-Stage Reaction; Two-Stage Reaction using Normal Amount of Membranes; Two-Stage Reaction using Low Concentration of Membranes. - [Read Protcol for In Vitro Endoplasmic Reticulum to Golgi Transport Reaction in Yeast]

Category: Protein Protocols: Protein Expression Protocols

Protein Expression Protocols and Applications Guide PDF. Promega. In vitro translation systems in Prokaryotes and Eukaryotes. Promega. - [Read Protein Expression Protocols and Applications Guide PDF]

Category: Signalling Signal Transduction Protocols

Protocol demonstrates that reactive astrocytes play a crucial role in wound healing and functional recovery by using mice with a selective deletion of the signal transducer and activator of transcription-3 (STAT3) or suppression of cytokine signaling-3 (SOCS3) under the control of Nestin gene promoter/enhancer (STAT3N–/–, SOCS3N–/–). Procedure includes: Surgical procedures, Functional evaluation, Immunohistochemistry, In vitro migration assay. - [Read Protocol for Conditional Ablation of stat3/socs3 Discloses the Dual Role for Reactive Astrocytes]

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

This protocol produces RNA transcripts from a cloned gene that can then be used in an in vitro translation assay. - [Read Protocol for In Vitro Transcription of RNA]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol for luciferase assay for in vitro detection. Protocol includes: Before cell lysate preparation; Cell lysate preparation; Protocol for manual luminometers; Protocol for plate reading luminometer; Preparation of protein assay reagent; Protein standards. - [Read Protocol for Luciferase Assay for In Vitro Detection]

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

Protocol describes an RNA Polymerase III (Pol III) transcription assay using an extract or proteins of choice. Pol III is the polymerase responsible for transcribing 5S RNA, tRNAs, and other small RNAs. α-Amanitin inhibits Pol II transcription in the assay. The newly-transcribed, radiolabeled RNA is visualized by autoradiography following Urea Polyacrylamide gel electrophoresis. - [Read Protocol for Polymerase III In Vitro Transcription]

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

RNA amplification by in vitro transcription protocol. Includes protocols on cDNA labeling and hybridization on cDNA microarrays. - [Read Protocol for RNA Amplification by In Vitro Transcription]

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

Protocol includes procedure for Transwell in vitro migration assays. - [Read Protocol for Trans-Well Migration Assay]

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

This protocol describes an in vitro reaction to assay mitotic spindle assembly. The assay uses Cytostatic Factor extract made from Xenopus eggs, fluorescently-labelled tubulin, and prepared sperm nuclei. Spindle assembly is monitored by immunofluorescence microscopy. - [Read Protocol Spindle Assembly In Vitro]

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

In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rRNA, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies. - [Read Protocol: Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters]

Category: Nucleic Acid Purification Protocols

Protocol for purification of in vitro synthesized mRNA with Microcon or Centricon Centrifugal Filters. - [Read Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters Protocol]

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]