transfection Protocols

Category: Reporter Gene Assays: GFP Assay Protocols

Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. These methods should be followed in the order presented, but any of them can be omitted when not needed. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Category: Microinjection Protocols

Fluorescence microscopy provides a powerful tool for imaging molecular components in living cells. Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Category: Avian Bird Protocols

Protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA). The dsRNA is injected into the spinal cord of the embryo. Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules. - [Read Injection of dsRNA and Electroporation in Avian Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA).  The dsRNA is injected into the spinal cord of the embryo.  Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules.  It may be necessary to optimize the stage of the embryo and the electroporation procedure to improve the effectiveness of in ovo RNAi—cell competence changes with differentiation. - [Read Injection of dsRNA and Electroporation in Avian Embryos Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This protocol introduces a method for maintaining mammalian cell culture suitable for treatment with siRNA. Transfection of cultured cells with siRNAs (see Transfection of siRNA Duplexes into Mammalian Cells) and downstream analysis of the knockdown cells. - [Read Mammalian Cell Culture and Preparation of Cells for RNAi in 24-Well Plates Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol introduces a method for maintaining mammalian cell culture suitable for treatment with siRNA.  Transfection of cultured cells with siRNAs and downstream analysis of the knockdown cells. - [Read Mammalian Cell Culture and Preparation of Cells for RNAi in 24-Well Plates Protocol]

Category: Transfection Protocols: Transient Transfection Protocols

Transient transfection of genes into naïve mouse T cells using nucleofection using a modified electroporation technique. This protocol offers the possibility for rapid functional in vivo studies of targeted genes in primary mouse T cells. - [Read Nucleofection and Adoptive Transfer of Primary Mouse T lymphocytes using Transient Transfection]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol for oligofection of small interfering RNAs in senescent human fibroblasts. This protocol allows the transfection of human senescent cells (regardless of the cause of cellular senescence) with an efficiency of around 70%. - [Read Oligofection of Small Interfering RNAs in Senescent Human Fibroblasts Protocol]

Category: Cell Biology and Cell Culture: Cell Electroporation Protocols

Optimization of Electroporation Transfection of DNA Protocol into Cell Lines. Eppendorf - [Read Optimization Protocol – Cell Line Electroporation PDF]

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: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes the preparation of polyethylenimine (PEI)/DNA nanoparticles for targeted gene delivery. This delivery strategy improves the efficiency of gene transfer by enhancing the entry of gene vectors into the desired cells and reducing uptake by nontarget cells. We describe here methods for the conjugation of targeting peptides to PEIs, formation of DNA complexes using the conjugated PEIs or nonconjugated PEIs together with targeting peptides, and cell transfection. - [Read PEI Nanoparticles for Targeted Gene Delivery Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

In preparation for FLIM-FRET analysis, the appropriate donor and acceptor components must be introduced into live or fixed cells. The method of introduction depends on the nature of the components and the state of the cells. For example, plasmid DNAs encoding a protein of interest fused to a variant of GFP may be introduced into live cells by transfection or microinjection, whereas labeled antibodies are delivered by microinjection. - [Read Probing Protein Interactions Using GFP and FRET Protocol]

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

Lipofectamine transient tranfection of mammalian cells in IMDM cell media. Julie B. Wolf, UMBC. - [Read Procedure for Transfection of Mammalian Cells Using Lipofectamine]

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

Two methods of screening pools of cloned cDNAs are discussed: transfection into mammalian cells and injection into Xenopus oocytes. - [Read Screening cDNA Libraries Constructed in Eukaryotic Expression Vectors Protocol]

Category: RNAi Technology Protocols

Protocol for si-RNA transfection of mouse bone marrow progenitors by electroporation. - [Read si-RNA Transfection of Mouse Bone Marrow Progenitors by Electroporation Protocol]

Category: RNAi Technology Protocols

Protocol Involves the transfection of siRNA into RAW 264. 7 cells using Lipofectamine 2000.  Cells are transfected with siRNA twice (on subsequent days).  Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection. - [Read siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine Protocol]

Category: RNAi Technology Protocols

Protocol Involves the transfection of siRNA into RAW 264. 7 cells using Lipofectamine 2000.  Cells are transfected with siRNA twice (on subsequent days).  Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection. - [Read siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine Protocol II]

Category: siRNA and RNAi Protocols

siRNA protocols - siRNA transfection. Mellor Lab - [Read siRNA protocols - siRNA transfection]

Category: siRNA and RNAi Protocols

siRNA Transfection Protocol. - [Read siRNA Transfection Protocol]

Category: siRNA and RNAi Protocols

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

Category: Transfection Protocols: Stable Transfection Protocols

This stage of the procedure describes the transfection with target genes of cell lines already expressing inducible tTA. In this example, the target genes are transfected on a plasmid that carries puromycin resistance as a selectable marker. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol II]

Category: siRNA and RNAi Protocols

Selection of siRNA duplexes from the target mRNA sequence, Preparation of siRNA duplexes, Useful siRNA reagent combinations, Transfection of siRNA duplexes, Sequences of siRNA duplexes used in our studies. Tuschl Lab. - [Read The siRNA user guide]

Category: RNAi Technology Protocols

The siRNA user guide. Includes: Selection of siRNA duplexes from the target mRNA sequence; Preparation of siRNA duplexes; Useful siRNA reagent combinations; Transfection of siRNA duplexes; Sequences of siRNA duplexes used in our studies. - [Read The siRNA User Guide]

Category: Transfection Protocols

The following procedure is for simultaneous transfection and plating of RAW 264.7 cells. This protocol results in approximately 50% to 70% cell viability, and of those viable cells, 20% to 40% are transfected when using pEYFP-N1 from Clontech. Include: Procedure for Splitting Cells before Transfection; Procedure for Preparing Lipofectamine 2000 and DNA; Preparation of RAW 264.7 Cells for Transfection. - [Read Transfecting and Plating RAW 264.7 Cells with Lipofectamine 2000 Protocol]