Assay:Single Round of In Vitro Transcription from Assembled Chromatin Templates Using a HeLa Cell Ex This protocol describes an in vitro transcription assay that allows for a single round of transcription from in vitro assembled chromatin. Comparing the activity of a receptor or transcriptional coactivator in an assay that measures only a single round of transcription with the results from multiple rounds of transcription can help elucidate the mechanism of transcriptional activation by those factors.
Basic Information on In Vitro Transcription The ability to synthesize RNA in the lab is critical to many techniques.Radiolabeled and nonisotopically labeled RNA probes, generated in small scale transcription reactions can be used in blot hybridizations and nuclease protection assays. This article includes information on: Requirements For Transcription, RNA Phage Polymerases, Template Options: Plasmids, PCR Products, Oligonuclotides and cDNA, Sense or Antisense, Conventional Or Large Scale Synthesis, Products for In Vitro Transcription.
Eukaryotic In Vitro Translation Systems Provides information on specific parameters you need to be aware of when using eukaryotic in vitro translation systems. Includes: DNA Template Considerations; Protein Labeling; Non-Radioactive Protein Labeling.
In Vitro Transcription Assay (Run-off Assay) using Permeabilized Cells 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
In Vitro Transcription With Yeast Nuclear Extract Protocol for in vitro transcription with yeast nuclear extract. Includes recipes for: 5x Acetate transcription buffer; Creatine phospho kinase; Phospho creatine; Stop mix; HA + 0.1 M potassium acetate; 5x glutamate transcription buffer (5 ml). Includes also protocol for Primer Extension Assay.
In vitro Translation Assays for CFTR Protocol outlines the general procedure and requirements for in vitro translation of CFTR and outlines some assays using in vitro translated product. Core glycosylation of CFTR occurs in the ER. An assay for this processing step requires the
addition of microsomal membranes to the basic in vitro translation mixture. This protocol takes this into account.
Nuclear Extract in vitro Transcription System 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.
Preparation of KC Nuclear Extract for In Vitro Splicing 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.
Protocol for Polymerase III In Vitro Transcription 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. Î±-Amanitin inhibits Pol II transcription in the assay. The newly-transcribed, radiolabeled RNA is visualized by autoradiography following Urea Polyacrylamide gel electrophoresis.
Protocol for Transcriptional Run- On Assays In this protocol nuclei isolated from cells expressing the gene of interest are incubated with radiolabeled UTP which is incorporated into nascent RNA transcripts by RNA polymerase molecules that were actively transcribing at the time the cells were harvested. Because very little denovo initiation of RNA synthesis occurs in isolated nuclei, transcription of the target gene can be measured by hybridizing the radiolabeled RNA to an excess of the target gene immobilized on a nitrocellulose or nylon
Protocol Spindle Assembly In Vitro 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.
Protocol: Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters 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.
Riboprobe In Vitro Transcription Systems 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.
Run- On Transcription Protocol Run-on transcription monitors the regulation of transcription in isolated nuclei. Protocol includes: Preparation of Nuclei, Run-On Transcription Reaction, Alkaline Hydrolysis of Run-Off Transcripts, Preparation of Filters and Hybridization. Solutions included: DNase Solution, Hybridization Mix, Denhardt Solution (100X) etc.
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