cosmid 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: DNA Protocols: Cosmid Library Protocols

Amplification of cosmid libraries may result in distorted representation of cloned genomic sequences and should be avoided wherever possible. If amplification should become necessary, it is best to expand the library by growth in liquid medium. - [Read Amplification and Storage of a Cosmid Library: Amplification in Liquid Culture 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: 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: DNA Protocols: DNA Sequencing Protocols: Big Dye Protocols

Big Dye protocols and notes for: cosmid, BAC, BAC, Fosmid Templates. - [Read Big Dye Protocols and Notes - Cosmid, BAC, BAC, Fosmid Templates]

Category: DNA Protocols: Cosmid Library Protocols

Protocol describes how to construct a library of 35-45-kb fragments of genomic DNA in the double cos site cosmid vector, SuperCos-1. The steps include: Linearization and dephosphorylation of SuperCos-1 DNA; Partial digestion of high-molecular-weight DNA with MboI; Dephosphorylation of high-molecular-weight genomic DNA; Ligation of cosmid arms to genomic DNA: Packaging and plating recombinants; Isolation and analysis of recombinant cosmids: Validation of the library. - [Read Construction of Genomic DNA Libraries in Cosmid Vectors Protocol]

Category: BACs Bacterial Artificial Chromosome Protocols

Sequencing conditions tested for the ABI Big-Dye terminators (PE-ABI #4303150 for the 1000 reaction kit - Description: TF,KIT BTD RR-1000) with various templates. Important to quantitate all templates by agarose gel electrophoresis vs size and concentration standards and do a few tests with different template concentrations to determine the optimal conditions for your reactions. Several conditions are given. - [Read Dye Protocols and Notes for Cosmid, BAC, BAC, Fosmid Templates]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for fluorescence in situ hybridization (FISH) on human chromosomes and interphase nuclei. Includes: Plasmid probes; COSMID-, YAC- and LIBRARY PROBES; Probe detection. - [Read Fluorescence In Situ Hybridization (FISH) on Human Chromosomes and Interphase Nuclei Protocol]

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Restriction Digestion Methods. Donis-Kellor lab manual. Restriction Enzyme Digests, Restriction Enzyme Buffers, Restriction Digestion of Plasmid, Cosmid, and Phage DNAs. - [Read Restriction Digestion Methods]

Category: DNA Protocols: Cosmid Library Protocols

Unamplified cosmid libraries are plated at high density onto 150-mm nitrocellulose or nylon filters and screened by hybridization - [Read Screening an Unamplified Cosmid Library by Hybridization: Plating the Library onto Filters Protocol]