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A Rapid Method for Generating Gene Deletions in Aspergillus fumigatus Protocol - http://www.aspergillus.org.uk/indexhome.htm?secure/laboratory_protocols/./genedel.html~main

Category: Fungi Protocols: Aspergillus Protocols

The technique makes use of an Escherichia coli strain expressing the redΑßΓ operon under the control of an inducible promoter. This enables the strain to carry out homologous recombination with only 50-60 bp of homologous sequence. The procedure does not require any DNA ligation and is very rapid. It allows a single gene or region on a cosmid to be replaced by a bi-functional selectable marker (having both an E. coli and an A. fumigatus marker). - [Read A Rapid Method for Generating Gene Deletions in Aspergillus fumigatus Protocol]

Bidirectional Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4139

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for bidirectional, blunt-end cloning of DNA fragments. The target DNA is PCR amplified and 3'-extensions are polished with Pfu DNA polymerase. The amplicon is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Bidirectional Cloning of PCR Products Protocol]

Directional Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4140

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for directional blunt-end cloning of DNA fragments. The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA. The products of the ligation reaction are used to transform competent Escherichia coli. A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Directional Cloning of PCR Products Protocol]

Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol - http://www.biologicalprocedures.com/bpo/arts/1/123/m123.pdf

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for the generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. - [Read Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol]

Hydroxylamine Mutagenesis of Plasmid DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4156

Category: Yeast Protocols: Yeast Nucleic Acid Protocols

Protocol produces mutagenized DNA that can be used for transformation of either Escherichia coli or yeast. - [Read Hydroxylamine Mutagenesis of Plasmid DNA Protocol]

Introduction of Double-Stranded RNA in C. elegans by Feeding Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4317

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

Protocol describes an easily scalable way of introducing double-stranded RNA (dsRNA) in Caenorhabditis elegans: feeding the nematode with bacteria that express dsRNA. When using an RNase-III-negative Escherichia coli strain (HT115), the efficiency of this method is comparable to the alternative. - [Read Introduction of Double-Stranded RNA in C. elegans by Feeding Protocol]

Introduction of Double-Stranded RNA in C. elegans by Feeding Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4317

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Protocol describes an easily scalable way of introducing double-stranded RNA (dsRNA) in Caenorhabditis elegans: feeding the nematode with bacteria that express dsRNA. When using an Rnase-III-negative Escherichia coli strain (HT115), the efficiency of this method is comparable to the alternative. - [Read Introduction of Double-Stranded RNA in C. elegans by Feeding Protocol]

Isolation of Plasmid DNA from Yeast Cells: A Ten-Minute Preparation - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4150

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast DNA Isolation Protocols

Protocol describes the purification release of plasmid DNA from yeast cells. The resulting plasmid DNA can be used to transform Escherichia coli or yeast. - [Read Isolation of Plasmid DNA from Yeast Cells: A Ten-Minute Preparation]

Maintenance of Probes in Bacteria Including Escherichia coli Protocol - http://wheat.pw.usda.gov/~lazo/methods/lazo/bactstor.html

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

Plasmid (pUC series) containing genomic DNA fragments are maintained in E. coli strain DH5aTM. The E. coli cultures are routinely cultured at 37 C on Luria-Bertani (LB) agar on or in LB broth containing Ampicillin (30 µg/ml) or Carbenicillin (50 µg/ml broth, 100 µg/ml agar). E. coli strains are usually preserved in stab agar or glycerol for mid-term storage and lyophilized for long-term storage. - [Read Maintenance of Probes in Bacteria Including Escherichia coli Protocol]

Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in E coli Protocol - http://www.plantmethods.com/content/1/1/6

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]

Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli - http://www.plantmethods.com/content/1/1/6

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]

Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli - http://www.plantmethods.com/content/1/1/6

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]

Purification of Bovine Lens and Bacterially Expressed Human Vimentin Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4675

Category: Cytoskeleton Protocols: Intermediate Filaments Protocols

Intermediate filaments (IF) are major cytoskeletal systems of vertebrate and many nonvertebrate cells whose expression is cell-type specific and developmentally regulated. This protocol describes a method for purifying one type of IF, vimentin, from bovine lens tissue. Purification of human vimentin expressed in Escherichia coli is also described. These methods are useful in the preparation of other IF protein subunits for microinjection studies as well. - [Read Purification of Bovine Lens and Bacterially Expressed Human Vimentin Protocol]

Transformation of Agrobacterium Using Electroporation Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4665

Category: Bacteria Transformation Protocols

Protocol describes a method for transforming Agrobacterium with plasmid DNA using electroporation in a manner similar to that commonly used for Escherichia coli. Although the transformation efficiency for Agrobacterium is lower than that for E. coli, it is possible to obtain adequate numbers of Agrobacterium transformants with this technique. - [Read Transformation of Agrobacterium Using Electroporation Protocol]

Transformation of Agrobacterium Using the Freeze-Thaw Method Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4666

Category: Bacteria Transformation Protocols

Protocol describes a method for transforming Agrobacterium with plasmid DNA using a freeze-thaw technique. Although the transformation efficiency for Agrobacterium is lower than that for Escherichia coli, it is possible to obtain adequate numbers of transformants with this technique. - [Read Transformation of Agrobacterium Using the Freeze-Thaw Method Protocol]

Articles (1-1 of 1)

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.