vectors Protocols

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol describes typical methods that are used to propagate and purify AAV vectors for experiments both in vitro and in vivo. Includes: Principles of the Triple Plasmid Transfection System; Plasmids; Transfection and Extraction of Virus; Purification of the AAV vector. - [Read A Protocol for AAV Vector Production and Purification]

Category: Cloning Protocols: Vector Protocols

Protocol is used to propagate and purify AAV vectors for experiments both in vitro and in vivo. Includes: Principles of the Triple Plasmid Transfection System; Plasmids; Transfection and Extraction of Virus; Purification of the AAV vector. - [Read AAV Vector Production and Purification 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: BACs Bacterial Artificial Chromosome Protocols

Information for BACs, Bacterial Artificial Chromosomes. Includes: Existing vectors; pBAC108L; pBeloBAC11; pBACe3.6; pFW11 and F' factor; pCC1BAC; pSMART VC Vectors; pIndigoBAC-5; Cosmids; pWEB-TNCTM; SuperCos 1; pFos1; PAC vectors; BAC purification; Putting the BAC into E. coli. - [Read BACs Bacterial Artificial Chromosomes]

Category: Cloning Protocols: Vector Protocols

Protocol for blunt-ended cloning into plasmid vectors. - [Read Blunt-ended Cloning into Plasmid Vectors Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol describes three standard methods to construct bacteriophage M13 recombinants: (1) ligating insert DNA to a linearized vector, prepared by cleavage of M13 RF with a single restriction enzyme; (2) using alkaline phosphatase to suppress self-ligation of the linearized vector, and (3) using M13 RF cleaved with two restriction enzymes for directional cloning. - [Read Cloning into Bacteriophage M13 Vectors Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Pairs of oligonucleotide primers used in PCR are often designed with restriction sites in their 5' regions.  In many cases, the sites are different in the two primers.  In this case, amplification generates a target fragment whose termini now carry new restriction sites that can be used for directional cloning into plasmid vectors.  The purified fragment and the vector are digested with the appropriate restriction enzymes, ligated together, and transformed into E. coli. - [Read Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA Protocol]

Category: PCR Protocols: PCR Cloning Protocols

This method of direct cloning takes advantage of the unpaired adenosyl residue added to the 3' terminus of amplified DNAs by Taq and other thermostable polymerases. - [Read Cloning PCR Products into T Vectors Protocol]

Category: Cloning Protocols: Vector Protocols

Protocol for cloning siRNA duplexes into pSUPER or pSUPER retro vectors. - [Read Cloning siRNA Duplexes into pSUPER or pSUPER Retro Vectors Protocol]

Category: RNAi Technology Protocols

Protocol for cloning siRNA duplexes into pSUPER or pSUPERretro vectors. - [Read Cloning siRNA Duplexes into pSUPER or pSUPERretro Vectors Protocol]

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Nice protocol for the preparation of Bacterial cells to allow uptake of plasmid vectors. Dr Peter Kille. Hot Metals Cardiff. - [Read Competent Cell Preparation [CaCl2]]

Category: DNA Protocols: Genomic DNA Library Protocols

This stage summarizes how to construct a cDNA library in a mammalian vector, using commercial kits. These kits are available from several manufacturers and generally include all the required reagents. - [Read Construction of cDNA Libraries in Eukaryotic Expression Vectors for Construction and Screening]

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: DNA Protocols: DNA Sequencing Protocols

Protocol for cycle sequence reactions for large insert plasmid templates. Includes: CycleSequenceConditions; Primers Useful For BAC and PAC Vectors. - [Read Cycle Sequence Reactions For Large Insert Plasmid Templates Protocol]

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

The goal of this method is to identify transcriptionally active genes in cloned segments of genomic DNA. The protocol uses hybridization and affinity purification to recover biotin-labeled cDNAs that bind to a 500-kb segment of human DNA cloned in a BAC vector. However, the method can be easily adapted to other clones of genomic DNAs cloned in high-capacity vectors. - [Read Direct Selection of cDNAs with Large Genomic DNA Clones Protocol]

Category: Cloning Protocols: Vector Protocols

Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes that generate incompatible termini and that the fragment of DNA to be cloned carries termini that are compatible with those of the doubly cleaved vector. - [Read Directional Cloning into Plasmid Vectors Protocol]

Category: Molecular Biology Protocols: DNA Ligation Protocols

Insertion of DNA into Plasmid Vectors, Insertion of DNA into l Phage Vectors, Self-Circularization of Linear DNA (Intramolecular Ligation), Linker (Adaptor) Ligation. Alam's Lab Univ Hawaii. - [Read DNA Ligation Protocol from Takara Shuzo Co.]

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

TRANSFECTION WITH FUGENE 6 Protocol and method. Johan Peränen Biocenter Helsinki. - [Read FuGene-6 Transfection Protocol PDF]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

The procedures involve the isolation and growth of primary cell cultures from rodent and human tissue as well as the use of viral vectors for the introduction and expression of mammalian genes in cells in culture and in live rodents. - [Read Growth of Primary Cell Culture and Viral Vector Handling Protocols]

Category: Bacteria Genetics Protocols

Protocol for the identification of positive GATEWAY expression clones when both the pENTRY and pDEST vectors contain the same marker for bacterial selection. Protocol describes ways in which difficult vector combinations can be used effectively to obtain the appropriate expression clone without having to convert the pENTRY clone or pDEST clone to vectors with compatible antibiotic resistances. - [Read Identification of Positive GATEWAY Expression Clones Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Protocol describes the use of vectorette PCR and single-site PCR to amplify the terminal sequences of genomic sequences cloned in high-capacity vectors such as PACs and YACs. - [Read Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors]

Category: Mouse Protocols: Mouse Cell Culture Protocols

The procedures involve the isolation and growth of primary cell cultures from rodent and human tissue as well as the use of viral vectors for the introduction and expression of mammalian genes in cells in culture and in live rodents. - [Read Isolation and Growth of Primary Cell Cultures from Mouse Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Method of choice when large amounts of mammalian DNA are required, for example, for Southern blotting (Rapid Isolation of Mammalian DNA, Rapid Isolation of Yeast DNA, Southern Blotting: Capillary Transfer of DNA to Membranes) or for construction of genomic libraries in bacteriophage {lambda} vectors.  Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 x 107 cultured aneuploid mammalian cells (e.g., HeLa cells). - [Read Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol used chiefly to generate large stocks of double-stranded DNA of strains of M13 that are routinely used as cloning vectors. Large amounts of single-stranded DNA of an individual recombinant may occasionally be needed for specific purposes, e.g., to generate many preparations of a particular radiolabeled probe or to construct large numbers of site-directed mutants. - [Read Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol]