strain Protocols

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]

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: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

4 strains of E. coli are used in these studies: JM101 for M13 infection and isolation, XL1BMRF'for M13 or pUC-based DNA transformation, and ED8767 for cosmid DNA transformation. To maintain their respective F' episomes necessary for M13 viral infection, JM101 is streaked onto a M9 minimal media plate and XL1BMRF' is streaked onto an LB plate containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid.. - [Read Bacterial Cell Maintenance Protocol]

Category: Molecular Biology Protocols: Carbohydrate Protocols

o determine the relative amounts of LPS carbohydrates present in a given strain. The assay can be done on one set of samples and then scanned at the various wavelengths for reasonable data on the 3 types of sugars. HEXOSE ASSAY, 6-DEOXYHEXOSE ASSAY, HEPTOSE ASSAY. Hancock Laboratory. - [Read Carbohydrate Assays]

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Direct method for determining efficiency in yeast. The plating efficiency of a strain is a measure of the percentage of cells in a culture that are capable of forming colonies (colony forming . - [Read Determining Plating Efficiency in Yeast: Direct Method]

Category: Yeast Protocols: Yeast Two-Hybrid Systems Protocols

Protocol utilizes the Y190 yeast strain, that characteristically turns from white to red during growth due to ademutation. Baits were subcloned into the pAS1cyh2 vector, and Preys subcloned into pACT2. - [Read Directed Yeast Two Hybrid Screening for MEKK1 Interaction Partners Protocol]

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol describes methods for isolation of DNA from a strain of S. cerevisiae carrying a recombinant YAC. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. The method is suitable for preparing DNA that will be used for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that do not require intact high-molecular-weight DNA. - [Read Growth of S. cerevisiae and Preparation of DNA Protocol]

Category: Competent Cells Protocols

This is an easy and straightforward protocol that gives efficiencies of 106 to 107 cfu/mg of plasmid DNA.  A growth curve is required for each strain that is prepared.
- [Read High Efficiency FCC Preparation and Tx Protocol]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to determine whether a strain will undergo senescence. - [Read How to Determine Whether a Strain will Undergo Senescence]

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: 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: Mouse Protocols: Mouse Reproductive System Surgery Protocols

Protocol describes a method for Caesarean section and fostering. Caesarean section is required if the recipient of an embryo transfer or any pregnant mouse has not given birth by the delivery time normal for the particular strain. - [Read Mouse Caesarean Section and Fostering Protocol]

Category: Molecular Model Organisms: Mouse Animal Protocols: Mouse Genotyping Protocols

PCR GENOTYPING PROTOCOL. 1) Cut toes of mice at approx. ten days of age and record sex, color, and strain. ... Back to Mouse Genotyping Resources. - [Read PCR GENOTYPING PROTOCOL]

Category: Yeast Protocols: Yeast Genetics Protocols

When more than one bait will be used to screen a single library, significant time and resources can be saved by performing the interactor hunt by interaction mating. In this protocol one strain is transformed with library DNA and the transformants are collected and frozen in aliquots. - [Read Performing a Hunt by Interaction Mating Protocol]

Category: Mouse Protocols: Mouse Embryology Protocols

Considerations in planning an effective mouse colony for transgenic production. Includes: Mouse strain; Colony size; Efficiency of superovulation; Parental Suitability; Pseudo-pregnancy. - [Read Planning an Effective Mouse Colony]

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: Fungi Protocols: Aspergillus Protocols

RAPD is a procedure for typing and fingerprinting isolates of a species. It can be used for epidemiological studies, such as investigations into hospital outbreaks and as a laboratory aid to keep track of cultures and to verify that mutants generated in the laboratory are genetically identical to the parental strain. In our hands, the use of one primer, R108, is sufficiently discriminatory to distinguish between the isolates of different strains. - [Read Random Amplification of Polymorphic DNA (RAPD) Typing and Fingerprinting Protocol]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

This method is advantageous for saving the occasional cultures that become contaminated. Yeast contaminated cultures will appear cloudy when slightly shaken and lymphocytes will not cluster together as much as normal. If cultures are suspect, a drop of culture can be streaked on a YPD media plate to check for growth of yeast colonies, or a 5 ml sample can be taken to Barnes Diagnostic Center for identification of yeast strain. - [Read Removal of Yeast Contamination from Lymphoblast Cultures Protocol]

Category: Yeast Protocols: Yeast Genetics Protocols: Yeast Replication Protocols

Protocol for replication timing using transient hemimethylation. Includes: Strain construction; Protocol for methylation timing; Quantitation. - [Read Replication Timing Using Transient Hemimethylation 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: Plant Biology Protocols: Arabidopsis Transformation Protocols

Agrobacterium tumefaciens strain transformation of arabidopsis thaliana. Steve Clough and Andrew Bent, University of Illinois at Urbana-Champaign. - [Read Simplified Arabidopsis Transformation Protocol]

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Protocol for spore germination. This procedure is typically used for the isolation and preparation of spores from a diploid strain heterozygous for a marked disruption (e.g., yfg1::his3+) Inoculation of the spore population into minimal medium lacking the nutritional supplement corresponding to the disruption marker (e.g., minimal medium lacking histidine) allows only the disruption spores to germinate. - [Read Spore Germination Protocol]

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

Some yeast strains are unstable (e. g., small YAC-bearing strains) and need to be repurified by streaking on an agar plate and then verifying the genetic content of the isolated colony before proceeding. In cases where the strain is unstable, plan to streak the cells onto the selective medium to retain the desired stock, (however, most strains can be streaked onto the complete medium, YPD). - [Read Streaking Yeast Stocks Protocols]