coli Protocols

Category: E Coli Protocols: E coli Transformation Protocols

Protocol is is used to prepare batches of competent bacteria that yield 5 x 106 to 2 x 107 transformed colonies/µg of supercoiled plasmid DNA. - [Read Preparation and Transformation of Competent E. coli Using Calcium Chloride Protocol]

Category: Bacteria Transformation Protocols

Protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/µg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C. - [Read Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells Protocol]

Category: E Coli Protocols: E coli Transformation Protocols

Protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/µg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C. - [Read Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells Protocol]

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Simple E.coli competent cell Protocol. Based on a protocol from Neil Olszewski and Scott Medberry.. Craig D. Amundsen - [Read Preparation of Competent Bacterial Cells]

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Preparation of Electrocompetent E. coli (i.e. DH5 ). PDF. Bjorkman Group, California I.T. - [Read Preparation of Electrocompetent E. coli (i.e. DH5 ). PDF.]

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol for the preparation of electrocompetent E. coli (i.e. DH5 ). - [Read Preparation of Electrocompetent E. coli DH5 Protocol]

Category: E Coli Protocols: E coli Transformation Protocols

Protocol for the preparation of electrocompetent E. coli. - [Read Preparation of Electrocompetent E. coli Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating.  This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). - [Read Preparation of Plasmid DNA by Large-scale Boiling Lysis Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating.  This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). - [Read Preparation of Plasmid DNA by Small-scale Boiling Lysis Protocol]

Category: C. elegans Protocols: C. elegans Cell Culture Protocols

Protocol describes how to seed plates with E. coli OP50 for C. elegans cultures. Includes: Bacterial Stock Plate Preparation; Bacterial Broth Preparation; Seeding Bacteria Plates for Culturing Worms. - [Read Preparation of Seeded NGM Plates For Worm Food Protocol]

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Preparation of ultra-competent E. coli cells for transformation. Dr. Chun-Ming Liu. Based on Inoue et al (1990). - [Read Preparation of ultra-competent E. coli cells for transformation]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Single-stranded templates of bacteriophage M13 DNA containing 20-30 residues of uracil in place of thymine are generated during growth of the bacteriophage in an F' strain of E. coli carrying mutations in the ung and dut genes. This DNA is used as a template in the Kunkel method of oligonucleotide-directed mutagenesis (Oligonucleotide-directed Mutagenesis of Single-stranded DNA). - [Read Preparation of Uracil-containing Single-stranded Bacteriophage M13 DNA Protocol]

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Detailed protocol Preparation and Steps for E.Coli Competent Cells procedure. Open Net Ware. - [Read Preparing Chemically Competent Cells]

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol for the transformation of e. coli by electroporation. - [Read Protocol Transformation of E. coli by Electroporation]

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]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the purification of GST-fusion proteins E. coli. (pGEX vector). - [Read Purification of GST-fusion proteins E. coli. (pGEX vector) Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP.  After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. - [Read Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol]

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

This method is used to isolate genomic yeast DNA or shuttle plasmids that replicate in both S. cerevisiae and E. coli. The DNA can be used as a template for PCR and for transformation. - [Read Rapid Isolation of Yeast DNA Protocol]

Category: Antibody Protocols: Antibody Production Protocols

This protocol describes how antibodies that react with bacterial-encoded proteins may be removed from polyclonal antisera by incubation with a bacterial lysate. - [Read Removal of Cross-reactive Antibodies from Antiserum: Incubation with E. coli Lysate]

Category: Antibody Protocols: Antibody Production Protocols

Antibodies directed against bacterial and phage-encoded proteins can be removed from sera by incubation with nitrocellulose filters impregnated with lysates of uninfected or phage-infected E. coli. - [Read Removal of Cross-reactive Antibodies from Antiserum: Pseudoscreening Protocol]

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

An expression library constructed in a bacteriophage {lambda} vector is plated on an appropriate E. coli strain in the absence of isopropylthio-ß-D-galactoside (IPTG). After 2-4 hours, the plates are moved to 37°C (to stabilize any fusion proteins that are temperature sensitive), and filters impregnated with IPTG are laid on top of the developing plaques. - [Read Screening Expression Libraries Constructed in Bacteriophage Lambda Vectors Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

A synthetic oligonucleotide annealed to single-stranded DNA derived from a recombinant bacteriophage M13 or phagemid template is used to prime the synthesis of complementary radiolabeled DNA. Synthesis is catalyzed by the Klenow fragment of E. coli DNA polymerase I, which extends the annealed primer for various distances along the single-stranded template DNA. - [Read Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates Protocol]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for systematic mutagenesis of E.coli K-12 MG1655 ORFs. Includes: Mutagenesis Project Progress; Construction of Sce-poson mutants; Mutagenesis of Essential Genes; Gene Gorging. - [Read Systematic Mutagenesis of E.coli K-12 MG1655 ORFs Protocol]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for systematic mutagenesis of e. coli K-12 MG 1655. - [Read Systematic Mutagenesis of E.coli K-12 MG1655 ORFs Protocol]

Category: DNA Protocols: TOPO Cloning

Topo Cloning procedure for cloning of small amounts DNA fragments by PCR. Cloning the PCR fragment into the TOPO vector, transforming E. Coli cells, and using blue/white selection to determine transformants. Culture inoculation, Qiagen Plasmid Prep protocol for plasmid isolation of the plasmid containing the cloned copies. Preuss Lab, Univ. Chicago. - [Read Topo Cloning Procedure]