constructs Protocols

Category: C. elegans Protocols: C. elegans Genetics Protocols

The cosuppression effect in C. elegans does not spread throughout the animal. Cosuppression in C. elegans can be triggered by highly repetitive transgenes that contain gene constructs. - [Read Cosuppression in C. elegans Protocol]

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Cosuppression is a process in Caenorhabditis elegans that closely resembles RNAi.  In contrast to RNAi, however, the cosuppression effect in C. elegans does not spread throughout the animal.  Cosuppression in C. elegans can be triggered by highly repetitive transgenes that contain gene constructs. - [Read Cosuppression in C. elegans Protocol]

Category: Mouse Protocols: Mouse Genetics Protocols

Protocol describes a method for electroporating DNA into ES cells, as well as selection methods. Pilot studies should be performed to optimize the conditions for each DNA construct. The selection method described here is one of the most complex. It involves targeting constructs in which the bacterial neomycin-resistance gene disrupts the coding sequence of the mouse gene. - [Read Electroporating DNA into Embryonic Stem (ES) Cells and Selection Methods Protocol]

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: Cloning Protocols

Protocol for ligating plasmid and target DNAs in low-melting-temperature agarose. Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. - [Read Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Protocol]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli. Describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in E coli Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

Describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli. Includes: Key steps in the EL250 RED-HR locus rescue and engineering procedure; Primer design and plasmid constructs; AtSTM gap-repair construct; Targeting construct backbone; Preparation of electrocompetent EL250 cells; Transformation of BAC F24o1 and induction of recombinogenic function in EL250; AtSTM locus rescue from BAC F24o1 by gap-repair HR. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli]

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: 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: Plant Biology Protocols: Plant Genetics Protocols

This procedure, which uses a root transformation protocol, provides a rapid method for assessing gene expression in Arabidopsis roots. It is useful for testing promoter:reporter gene constructs, for expressing genes, the overexpression of which is lethal in whole plants, and for transforming the roots of plants that are recalcitrant to conventional transformation techniques. The protocol has been used successfully with Ws, No-0, and RLD ecotypes. - [Read Transgene Expression in Regenerated Roots]