linearized Protocols

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: Molecular Biology Protocols: DNA Ligation Protocols

DNA ligations are performed by incubating DNA insert with linearized cloning vector in the presence of ligation buffer, rATP, and T4 DNA ligase. Roe. Univ. Oklahoma. - [Read DNA ligation with Linearized DNA]

Category: Cloning Protocols: Vector Protocols

To minimize self-ligated vector in your transformation, treat your linearized vector with a phosphatase to remove the 5' phosphates necessary for ligation. This should improve the percentage of colonies with inserts. - [Read Phosphatase Treatment of Linearized Vector Protocol]

Category: Avian Bird Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes. The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII). The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. After purification, the RNA strands are annealed to yield a dsRNA molecule suitable for RNAi in avian embryos. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes.  The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII).  The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes how to prepare double-stranded RNA (dsRNA) for RNA interference in Drosophila by synthesis of individual RNA strands from linearized plasmid templates, followed by annealing of the strands.  DsRNA molecules with a length of 500-800 bp seem to be most active.  The dsRNA can be made from cDNA or genomic DNA templates, as long as most of the dsRNA corresponds to presumptive exon sequence. - [Read Synthesis of dsRNA for RNAi in Drosophila: Plasmid Template Method Protocol]