bacterial Protocols

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 alkali and SDS. - [Read Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation 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: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks. - [Read Preparation of Plasmid DNA: Toothpick Minipreparation 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: Viral Culture and Isolation Protocols: Viral Culture Protocols

High-titer stocks of bacteriophage {lambda} are easily prepared by infecting small-scale bacterial cultures. - [Read Preparing Stocks of Bacteriophage {lambda} by Small-scale Liquid Culture Protocol]

Category: Recombinant Protein Protocols

From plasmid to protein using bacterial expression. Transform appropriate DNA plasmid, Make a starter culture for protein expression, bacteria culture for protein expression. Sosnick Group Chicago. - [Read Protein Expression and Purification Protocol]

Category: Viral Protocols

Isopycnic centrifugation through CsCl gradients is used to prepare infectious bacteriophage {lambda} particles of the highest purity that are essentially free of contaminating bacterial nucleic acids. - [Read Purification of Bacteriophage lambda Particles by Isopycnic Centrifugation through CsCl Gradients]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Protocol describes a method for removing antibodies that react with bacterially encoded proteins by passing a crude preparation of immunoglobulins through a column containing immobilized bacterial proteins. - [Read Removal of Cross-reactive Antibodies from Antiserum: Affinity Chromatography 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: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for restriction endonuclease digestion of DNA in agarose plugs. Genomic DNA isolated from mammalian, yeast, or bacterial cells can be digested with restriction endonucleases by incubating agarose plugs containing the DNA in the presence of the desired enzyme.  After digestion, the DNA can be fractionated by pulsed-field gel electrophoresis and either isolated from the gel or analyzed by Southern Hybridization. - [Read Restriction Endonuclease Digestion of DNA in Agarose Plugs Protocol]

Category: Bacterial Protocols

Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. - [Read Screening Bacterial Colonies by Hybridization: Intermediate Numbers Protocol]

Category: Bacterial Protocols

This procedure is used to plate, replicate, and subsequently screen large numbers of bacterial colonies (up to 2 x 104 colonies per 150-mm plate or 104 colonies per 90-mm plate). - [Read Screening Bacterial Colonies by Hybridization: Large Numbers Protocol]

Category: Bacterial Protocols

Protocol used to screen a small number of bacterial colonies (<200) that are dispersed over several agar plates and are to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar plate. - [Read Screening Bacterial Colonies by Hybridization: Small Numbers Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Plaques formed by M13 bacteriophages or bacterial colonies transformed by plasmids carrying specific mutations can be detected by hybridization, using a radiolabeled oligonucleotide that forms a perfect duplex with the mutant sequence. Hybridization is carried out under conditions of low stringency that allow the radiolabeled oligonucleotide to anneal to both mutant and wild-type DNAs. - [Read Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligos]

Category: Protein Protocols: Protein Precipitation Procols

Simple and Reliable Method To Precipitate Proteins from Bacterial Culture Supernatant. Randolph B. Caldwell et al., AEM ASM. - [Read Simple and Reliable Method To Precipitate Proteins from Bacterial Culture Supernatant PDF]

Category: DNA Protocols: SNP Protocols

SNP detection with mutagenic primers. Input sequence will be searched to find changes in one nucleotide near the location of the SNP, so that mutagenic primers may be easily designed. Bikandi, J., San Millán, R., Rementeria, A., and Garaizar, J. In silico analysis of complete bacterial genomes: PCR, AFLP-PCR, and endonuclease restriction. Bioinformatics 2004 Mar 22;20(5):798-9. - [Read SNP detection with mutagenic primers]

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

Protocol describes a method for storing bacterial cultures containing glycerol which are growing in liquid media. - [Read Storage of Bacterial Cultures Growing in Liquid Media Protocol]

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

Protocol describes a method for storing bacterial cultures that are growing on solid media. It is useful for storing copies of cDNA. - [Read Storage of Bacterial Cultures Growing on Solid Medium Protocol]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

Protocol describes a target selective S. aureus whole cell assay that combines agar-diffusion and protein over expression techniques.  This agar based two-plate differential sensitivity assay was used to help confirm the newly discovered antibiotic platensimycin inhibited bacterial growth by specifically targeting the essential FASII enzyme FabF6. - [Read Target Specific Whole Cell Assay for Antibacterial Drug Discovery Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

When an individual bacterial virus grows in a bacterial host suspended in a top agar lawn, its progeny infect and lyse the surrounding host cells. This causes the appearance of a "hole" or plaque in the otherwise homogeneous bacterial lawn. Since each plaque represents a single virus, the number of viruses in the aliquot added to the plate is equal to the number of plaques which appear. - [Read Titering of Bacterial Viruses Protocol]

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]

Category: Plant Biology Protocols: Arabidopsis Transformation Protocols

A. tumefaciens is a soil-dwelling bacterium that transforms normal plant cells into tumor-forming cells by inserting a piece of bacterial DNA (the transfer, or "T," DNA) into the plant cell genome. The Ti plasmid also carries many of the transfer functions for mobilizing the T-DNA. This article provides a brief discussion of the principles of T-DNA transformation, including consideration of T-DNA vectors and their hosts. - [Read Vectors and Agrobacterium Hosts for Arabidopsis Transformation Protocol]

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol describes how to transform E. coli with BAC DNA by electroporation. - [Read Working with Bacterial Artificial Chromosomes Protocol]