construct Protocols

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol describes three standard methods to construct bacteriophage M13 recombinants: (1) ligating insert DNA to a linearized vector, prepared by cleavage of M13 RF with a single restriction enzyme; (2) using alkaline phosphatase to suppress self-ligation of the linearized vector, and (3) using M13 RF cleaved with two restriction enzymes for directional cloning. - [Read Cloning into Bacteriophage M13 Vectors Protocol]

Category: DNA Protocols: Genomic DNA Library Protocols

This stage summarizes how to construct a cDNA library in a mammalian vector, using commercial kits. These kits are available from several manufacturers and generally include all the required reagents. - [Read Construction of cDNA Libraries in Eukaryotic Expression Vectors for Construction and Screening]

Category: DNA Protocols: Cosmid Library Protocols

Protocol describes how to construct a library of 35-45-kb fragments of genomic DNA in the double cos site cosmid vector, SuperCos-1. The steps include: Linearization and dephosphorylation of SuperCos-1 DNA; Partial digestion of high-molecular-weight DNA with MboI; Dephosphorylation of high-molecular-weight genomic DNA; Ligation of cosmid arms to genomic DNA: Packaging and plating recombinants; Isolation and analysis of recombinant cosmids: Validation of the library. - [Read Construction of Genomic DNA Libraries in Cosmid Vectors 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: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

ES / MEF cell culture and electroporation of targeting construct. Gary Brown - [Read ES / MEF cell culture and electroporation of targeting construct]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: MEF Protocols Mouse Embryonic Fibroblasts

Dr. Gary Brown. Electroporation and transfection of ES / MEF cells using electroporation for a targeting construct. - [Read ES / MEF cell culture and electroporation of targeting construct]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Protocol for ES/MEF cell culture and electroporation of targeting construct. - [Read ES / MEF Cell Culture and Electroporation of Targeting Construct Protocol]

Category: Stem Cell Protocols: Mouse Embryonic Fibroblasts Protocols

Protocol for ES/MEF cell culture and electroporation of targeting construct. - [Read ES / MEF Cell Culture and Electroporation of Targeting Construct Protocol]

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

Protocol for ES / MEF cell culture and electroporation of targeting construct. - [Read ES / MEF Cell Culture and Electroporation of Targeting Construct Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol for ES / MEF cell culture and electroporation of targeting construct. - [Read ES / MEF Cell Culture and Electroporation of Targeting Construct Protocol]

Category: DNA Protocols: Genomic DNA Library Protocols

Protocol describes how to construct and amplify a genomic DNA library in a plasmid vector (pSPL3) equipped to express cloned exons in transfected mammalian cells. - [Read Exon Trapping and Amplification for Construction of the Library Protocol]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

The same GFP-tagged actin construct used in cell transfection experiments has been used to produce transgenic mice. Transgenic animals allow the imaging of brain tissue in the intact animal, as acutely cut slices or as organotypic slice cultures. They also serve as a source of cells for imaging neurons at high resolution in dispersed low-density cell culture. In contrast to cells transfected in culture, where the level of actin-GFP expression in neurons varies considerably, transgenic mice... - [Read Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol used chiefly to generate large stocks of double-stranded DNA of strains of M13 that are routinely used as cloning vectors. Large amounts of single-stranded DNA of an individual recombinant may occasionally be needed for specific purposes, e.g., to generate many preparations of a particular radiolabeled probe or to construct large numbers of site-directed mutants. - [Read Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

Protocol is used to establish conditions for restriction enzyme digestion of eukaryotic genomic DNA that will generate fragments of a size appropriate for construction of genomic libraries.  To construct a genomic library, the average length of the starting genomic DNA should be at least eight times the capacity of the vector. - [Read Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions Protocol]

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: Yeast Protocols: Yeast Two-Hybrid Systems Protocols

Protocol describes how to generate a plasmid construct (pBAIT) that expresses a target protein fused to the bacterial LexA protein. PBAIT is cotransformed into yeast with a lexAop-lacZ reporter plasmid carrying the bacterial lacZ gene under the control of the lexA operator. The recipient yeast strain contains a chromosomally integrated leu2 reporter gene, also under the control of the lexA operator. - [Read Two-hybrid Systems Stage 1: Characterization of a Bait-LexA Fusion Protein Protocol]