transfection Protocols

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons. The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. The calcium phosphate method can be used to transfect neurons that have already been growing on coverslips in vitro. Transfected cells suitable for imaging can be obtained in cultures up to 15 days in vitro. - [Read Transfecting Cultured Hippocampal Neurons with an Actin-GFP Plasmid]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols

Protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons.  The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. - [Read Transfecting Cultured Hippocampal Neurons with an Actin-GFP Plasmid Protocol]

Category: Transfection Protocols

DEAE-dextran is generally used to obtain a burst of transient expression of cloned genes after transfection of mammalian cells. Many variants of the technique have been described, all of which seek to maximize the uptake of DNA and to minimize the cytotoxic effects of DEAE-dextran. In this protocol cells are exposed briefly to a high concentration of DEAE-dextran-DNA and then to chloroquine diphosphate, which is a facilitator of transfection. - [Read Transfection Mediated by DEAE-Dextran: High-efficiency Method Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Transfection of primary leukocytes has traditionally been a challenging but much desired protocol. It allows not only the analysis of cells in a more natural state to a cell line system, it enables the direct comparison of, for e.g. transcriptional activity using luciferase reporters, in immune cells taken from genetically-altered mice. In addition, importantly it allows for "rescue experiments" in knockout cells & the ability to over-express or reconstitute wild-type and/or mutated constructs. - [Read Transfection of Bone Marrow-Derived Mast Cells for Transcription Factor Luciferase Reporter Assays]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol describes transfection of plasmid DNA into primary hippocampal neurons using DNA/calcium-phosphate (CaPO4) coprecipitation. The precise pH of the transfection medium and the incubation time of cells with the coprecipitate are critical for reproducible and efficient transfection. Once these parameters are optimized for a given plasmid, the method is easily adapted for transfection of other established cell lines. - [Read Transfection of Hippocampal Neurons with Plasmid DNA Using Calcium Phosphate Coprecipitation]

Category: Transfection Protocols

Transfection of IMEC cells using SuperFect Transfection Reagent protocol. Protocol includes experimental design considerations. - [Read Transfection of IMEC Cells Using SuperFect Transfection Reagent Protocol]

Category: Transfection Protocols

Protocol describes a method for the delivery of siRNAs into mammalian cells in the absence of reporter plasmids. This is best achieved with transfection reagents developed for the delivery of antisense oligodeoxynucleotides. The quantities of reagents given below are calculated for the transfection of one well of a 24-well plate. - [Read Transfection of Mammalian Cells with siRNA Duplexes Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol describes a method for the delivery of siRNAs into mammalian cells in the absence of reporter plasmids.  This is best achieved with transfection reagents developed for the delivery of antisense oligodeoxynucleotides.  The quantities of reagents given below are calculated for the transfection of one well of a 24-well plate. - [Read Transfection of Mammalian Cells with siRNA Duplexes Protocol]

Category: Cloning Protocols: Vector Protocols

Protocol for the transfection/Retroviral Infection pCL-Eco+Gene of interest into 293T cells infecting HC11 cells. Includes: Split 293T cells for transfection; Transfect 293T cells; Split HC11 cells for infection; Infect target (HC11) cells. - [Read Transfection/Retroviral Infection pCL-Eco+Gene of Interest into 293T Cells Infecting HC11 Cells]

Category: Transfection Protocols: Transient Transfection Protocols

Transient transfection into mammalian cells is a convenient way to over express and obtain protein expression. Protocol includes: Culture conditions; Transfection of experimental cells; Preparation of Mammalian Cell Lysate for Luciferase assay. - [Read Transient Transfection and Luciferase assay]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Transient transfection into mammalian cells is a convenient way to over express and obtain protein expression. Protocol includes: Culture conditions; Transfection of experimental cells; Preparation of Mammalian Cell Lysate for Luciferase assay. - [Read Transient Transfection and Luciferase Assay Protocol]

Category: Transfection Protocols: Transient Transfection Protocols

Protocol for transient transfection and spin infection. - [Read Transient Transfection and Spin Infection Protocol]

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

Transient transfection into 293T cells Protocol. transfection, calcium phosphate, overexpression, protein production. Jun Takagi - [Read Transient transfection into 293T cells]

Category: Transfection Protocols: Transient Transfection Protocols

Transient transfection into 293T cells is a convenient way to overexpress and obtain both cellular and extracellular (secreted or membrane) proteins. 293 is a human renal epithelial cell line which is transformed by adenovirus E1A gene product. 293T is a derivative which also express SV40 large T antigen, allowing episomal replication of plasmids containing the SV40 origin and early promoter region. They (both) have the unusual property of being highly transfectable. - [Read Transient Transfection Into 293T Cells Protocol]

Category: Transfection Protocols: Transient Transfection Protocols

Protocol for the transient transfection of COS cells. - [Read Transient Transfection of COS Cells Protocol]

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

Protocol optimized for Cos-1 cells. Successful transfection of each cell type requires optimization of the basic protocol. LIPOFECTAMINE method of transient transfection. Upstate. - [Read Transient Transfection of Cos-1 Cells]

Category: Transfection Protocols: Transient Transfection Protocols

Transient transfection protocol for the ecotropic/amphotropic producer line including titering and concentration of viral stocks. - [Read Transient Transfection Protocol: Viral Production]

Category: Transfection Protocols: Transient Transfection Protocols

This transient transfection protocol for the ecotropic/amphotropic producer line includes instructions for titering and concentration of viral stocks. This procedure gives consistently higher transfection efficiency. - [Read Transient Transfection: Viral Production]

Category: Protein Protocols: Protein Ubiquitination Protocols

Ultimate His-UB Assay for Mammalian Cells. Tansey Lab Protocols. William P. Tansey. PREPARATION OF NI-NTA-AGAROSE, HARVESTING THE TRANSFECTION., ELUTION OF HIS-TAGGED PROTEINS. - [Read Ultimate His-UB Assay for Mammalian Cells]