vectors Protocols

Category: Cloning Protocols

Protocol for ligating plasmid and target DNAs in low-melting-temperature agarose. Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. - [Read Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Protocol]

Category: Developmental Biology

Describe the use of replication-incompetent retroviral vectors for the analysis of lineal relationships in developing vertebrate tissues. An overview of the relevant aspects of the retroviral life cycle, and the strategies and current methods in use at their laboratory are described. - [Read Lineage Analysis Using Retroviral Vectors]

Category: Cloning Protocols: Vector Protocols

Protocol describes the use of replication-incompetent retroviral vectors for the analysis of lineal relationships in developing vertebrate tissues. - [Read Lineage Analysis Using Retroviral Vectors Protocol]

Category: RNA Protocols: cDNA Libraries Library

The Queensland Institute for Medical Research (QIMR) lab's method for automated sequencing for expressed sequence tags from Schistosoma japonicum and/or S. mansoni cDNA libraries constructed in the directional vectors lambda ZAP II and UNI ZAP XR. Paul Br - [Read Method for automated sequencing for expressed sequence tags from Schistosoma japonicum and/or S. man]

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes the preparation of polyethylenimine (PEI)/DNA nanoparticles for targeted gene delivery. This delivery strategy improves the efficiency of gene transfer by enhancing the entry of gene vectors into the desired cells and reducing uptake by nontarget cells. We describe here methods for the conjugation of targeting peptides to PEIs, formation of DNA complexes using the conjugated PEIs or nonconjugated PEIs together with targeting peptides, and cell transfection. - [Read PEI Nanoparticles for Targeted Gene Delivery Protocol]

Category: Cytogenetics Protocols

Details a placenta specific gene manipulation by transducing blastocysts with lentiviral vectors1. After a removal of zona pellucida which functions as a physical barrier, trophoblast cells lying outermost layer of blastocyst were transduced from outside with high-titer lentiviral vectors. As most placental cells descend from trophoblast cells while fetus originated from inner cell mass, transgene expression can be observed in trophoblast cells from preimplantation stages and in placenta... - [Read Placenta Specific Gene Manipulation by Transducing Zona-Free Blastocyst using Lentiviral Vector]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Bacteriophage {lambda} vectors that allow genetic selection of recombinant bacteriophages (e.g., the EMBL series, {lambda}2001, {lambda}DASH, {lambda}ZAP, and {lambda}gt10) can be prepared for cloning by simple digestion by one or more restriction enzymes. - [Read Preparation of Bacteriophage lambda DNA Cleaved with a Single Restriction Enzyme Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Many replacement vectors (e.g., the EMBL series, {lambda}2001, and {lambda}DASH) contain a series of restriction sites, arranged in opposite orientations, at each end of the central stuffer fragment. Digestion of these vectors with two different restriction enzymes yields left and right arms, a stuffer fragment, and short segments of the polycloning sites. These can easily be removed from the arms by differential precipitation with isopropanol or spun-column chromatography. - [Read Preparation of Bacteriophage lambda DNA Cleaved with Two Restriction Enzymes Protocol]

Category: Viral Manipulation Protocols: Phage Protocols

Protocol describes methods to superinfect bacteria carrying a recombinant phagemid with a high-titer stock of an appropriate helper virus and to assay the yield of filamentous virus particles that carry single-stranded copies of the phagemid DNA. The key to success in using phagemids is to prepare a stock of helper virus whose titer is accurately known. - [Read Producing Single-stranded DNA with Phagemid Vectors Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rRNA, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies. - [Read Protocol: Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant proteins, constructed in pGEX vectors, are fused to glutathione S-transferase (GST) and can be purified to near homogeneity by affinity chromatography on glutathione-agarose.  Bound GST-fusion proteins are readily displaced from the column by elution with buffers containing free glutathione. - [Read Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose Protocol]

Category: PCR Protocols: PCR Cloning Protocols

In this protocol sequences cloned in standard bacteriophage or plasmid vectors are amplified in PCRs containing primers targeted to flanking vector sequences.  The amplified fragments can be analyzed by gel electrophoresis, DNA sequencing, and/or restriction mapping.  Many colonies or plaques can be assayed simultaneously. - [Read Rapid Characterization of DNAs Cloned in Prokaryotic Vectors Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Protocol exploits the discovery that Rnase A can efficiently cleave at single rC or rU bases embedded in double-stranded DNA.  Entire plasmid vectors are amplified using long, high-fidelity PCR with riboprimers, which carry a single rC residue at their 3' end.  Target DNA is amplified using similar primers, which also end in a rC residue. - [Read Ribocloning: DNA Cloning and Gene Construction Using PCR Primers Terminated with a Ribonucleotide]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

Two methods of screening pools of cloned cDNAs are discussed: transfection into mammalian cells and injection into Xenopus oocytes. - [Read Screening cDNA Libraries Constructed in Eukaryotic Expression Vectors 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: DNA Protocols: cDNA Library Protocols

A cDNA library constructed in a plasmid expression vector of the pUC, pUR, or pEX series is plated on agar medium and then replicated onto filters, which are transferred to plates containing IPTG. After 2-4 hours of induction, the colonies are lysed with chloroform and then screened with appropriate antibodies. - [Read Screening Expression Libraries Constructed in Plasmid Vectors Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol describes a split luciferase complementation assay that can be used to repetitively and noninvasively study the interaction of proteins in small living animals. After the expression of the appropriate vectors has been checked in cell culture in vivo, studies can be performed either by implanting transiently transfected cells for short-term analysis (maximum of 7 days), or with tumor models grown from tumor cells stably expressing the complete reporter system. - [Read Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Living Mice]

Category: Tetrahymena Protozoa Protocols

Protocol describes a method for transformation of the Tetrahymena using electroporation. The vector is electroporated into cells after mating, where it is incorporated into the DNA of developing macronuclei. Because T. thermophila can be propagated indefinitely without conjugation, transformation of the macronucleus provides a way to obtain stable somatic transformants. DNA vectors transformed using this protocol include those containing drug-resistant versions of Tetrahymena genes. - [Read Transformation of Tetrahymena thermophila by Electroporation 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: Cloning Protocols: Vector Protocols

Information on how to handle viral vectors. Includes: Adenovirus;  MoMLV retrovirus; MoMLV-based Retroviral Vectors: Base classification BSL-1. - [Read Viral Vector Handling Information]