bacteria Protocols

Category: Microbiology: Bacterial E.Coli Culture Media & Plates

LB Agar: Luria Broth, X-Gal preparation, IPTG and ampicillin. Petri dish preparation. Pat Heslop-Harrison and Trude Schwarzacher, Univ. Leicester - [Read Agar Plates for Selection of Clones in Bacteria]

Category: Microbiology: Mini-Prep Plasmid Purification

Harvesting and Lysis of Bacteria. Mini-prep plasmid purification using Solution I, Solution II and Solution III. The Preuss Lab. Univ. Chicago. - [Read Alkaline Lysis Mini-Prep Protocol]

Category: DNA Protocols: Cosmid Library Protocols

Amplification of cosmid libraries may result in distorted representation of cloned genomic sequences and should be avoided wherever possible. In this method of amplification, distortion of the library is rarely a problem because at no stage are bacteria containing different recombinant cosmids grown in competition with one another. - [Read Amplification and Storage of a Cosmid Library: Amplification on Filters Protocol]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

This assay is used when working with phage vectors carrying the beta-gal gene. If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-gal gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta-gal will work as indicator host bacteria; a single chromosomal copy of the gene is not a problem. - [Read Assay for Phage Containing the Beta-galactosidase Gene]

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

This assay is used when working with phage vectors carrying the beta-galactosidase gene (often used for immunological screening). If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-galactosidase gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta- galactosidase will work as indicator host bacteria. - [Read Assay for Phage Containing the Beta-galactosidase Gene Protocol]

Category: C. elegans Protocols

Protocol for bacteria- mediated (feeding) RNAi. - [Read Bacteria-Mediated (Feeding) RNAi Protocol]

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

To 2mls of mid-log culture or 1ml of freshly saturated culture add 1ml(or an equal volume) of glycerol solution, mix gently, then freeze rapidly in liquid nitrogen or on dry ice. Store at -20°C and - 70°C. - [Read Bacterial Glycerol Stocks Protocol]

Category: PCR Protocols: Colony PCR

Protocol describes the use of PCR to screen for bacteria that carry recombinant plasmids.  The PCR can be carried out using the same primers as for amplification of the cloned insert.  To determine the orientation of the insert, a third, insert-specific primer that is asymmetrically distanced from the clonal insertion site can be used. - [Read Colony PCR Protocol II]

Category: DNA Protocols: Genomic DNA Library Protocols

Protocol for the construction of a Yeast genomic library. Includes: Prepare the genomic DNA; Prepare the Library Vector; Ligate the Digested Genomic DNA to the Digested Vector DNA; Prepare Library DNA from Bacteria. - [Read Construction of a Yeast Genomic Library Protocol]

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Frozen Competent E.Coli Cells. Methods and protocol for freezing E.Coli Bacteria Competent Cells in aliquots for later use re-use. (Inoue et al., 1990 Gene 96:23). Koshland. - [Read Frozen Competent E.Coli Cells]

Category: Staining Protocols: Gram Staining Protocols

Gram-positive and Gram-negative organisms form a complex of crystal violet and iodine within the bacterial cell during the Gram-staining procedure. Gm+ organisms are thought to resist decolorization by alcohol or acetone because cell wall permeability is markedly decreased when it is dehydrated by these solvents. Thus, the dye complex is entrapped within the cell, resist being washed out by the solvents, and Gm+ bacteria remain purple following this differential stain. - [Read Gram Staining Protocol]

Category: Laboratory Model Organisms Protocols

Protocol describes the growth and concentration of the alga Chlorogonium elongatum as a food source for culturing freshwater hypotrichs. Most freshwater hypotrichs (including Oxytricha nova, O. fallax, and O. trifallax; Euplotes aediculatus and E. eurystomous; and Stylonychia lemnae) can be grown to high density with Chlorogonium as the food organism. A similar regimen can be used to prepare other food sources such as Tetrahymena or bacteria (e.g., Aerobacter aerogenes). - [Read Growth and Concentration of Chlorogonium for Culturing Freshwater Hypotrichs Protocol]

Category: PCR Protocols

PCR polymerase costs can be high. If you are willing to work, you can produce bacteria containing the clone. It appears to produce lots of Taq and is quite stable. The proceedure takes 4 days start to (15 000 units of Taq) finish. The Taq also appears ver - [Read Home-made Taq Polymerase Purification]

Category: Western Blot Protocols: General Western Blot Methods Protocols

For immunoblotting experiments, it is often important to compare the total amount of an antigen from many different sources or to learn if a particular source has the antigen under study.  In the approach described here, tissue cultures, bacteria, yeast cells, tissues, and other sources of antigens are disrupted directly in an electrophoresis sample . - [Read Immunoblotting: Preparing Cell Lysates Protocol]

Category: Bacterial Cell Culture Protocols

In this protocol, bacterial cells are lysed by being subjected to short, intense treatments with ultrasound, which breaks the cell walls and shears the DNA into sizes that will not affect the viscosity of the samples. Note that this method causes some denaturation of the samples. The resulting lysate is ready for preclearing. - [Read Immunoprecipitation: Lysing Bacteria by Sonication Protocol]

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]

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]

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

Double-stranded RNA (dsRNA) can be introduced into Caenorhabditis elegans by soaking the animals in a solution of dsRNA. Alternative methods are dsRNA injection (see Introduction of Double-stranded RNA in C. elegans by Injection) and feeding the animals with bacteria that produce dsRNA. - [Read Introduction of Double-Stranded RNA in C. elegans by Soaking Protocol]

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Double-stranded RNA (dsRNA) can be introduced into Caenorhabditis elegans by soaking the animals in a solution of dsRNA.  Alternative methods are dsRNA injection and feeding the animals with bacteria that produce dsRNA. - [Read Introduction of Double-Stranded RNA in C. elegans by Soaking Protocol]

Category: Bacterial Protocols

Protocol was developed to isolate Wolbachia from adult Drosophila, but it can be adapted for other insects. In some insects leg removal prior to isolation facilitates hemolymph extrusion. - [Read Isolation of Live Bacteria from Adult Insects Protocol]

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

Protocol for liquid culture of worms. Includes: superbroth; S- basal; worm plates; GROWING THE BACTERIA (WORM FOOD); GROWING THE WORMS; AFTER THE CULTURE HAS GROWN; PREPARING EGGS TO START SYNCHRONIZED LIQUID CULTURES. - [Read Liquid Culture of Worms Protocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Caenorhabditis elegans, a small (adults are ~1 mm long), free-living soil nematode that feeds on bacteria, is an ideal organism for applying various live microscopy techniques. This protocol describes useful techniques for preparing C. elegans for live microscopic analysis. Details of sample preparation depend on the developmental stage of the worm to be studied. - [Read Live Imaging of Caenorhabditis elegans: Preparation of Samples]

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]

Category: Microbiology: Mini-Prep Plasmid Purification

Using Solutions A for Lysis, Solutions B for Neutralization Solution, and Solution C for washing. Dr.David Peyton. - [Read Mini-Prep Protocol for Purification of Plasmid DNA from Bacteria]

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

Protocol for plasmid midi-prep from bacteria. - [Read Plasmid Midi-Prep from Bacteria Protocol]