plasmid Protocols

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with alkali and SDS. - [Read Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating.  This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). - [Read Preparation of Plasmid DNA by Large-scale Boiling Lysis Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Large (>15 kb), closed circular plasmids are prepared (albeit inefficiently and in small yield) by lysing bacteria with SDS. - [Read Preparation of Plasmid DNA by Lysis with SDS Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating.  This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). - [Read Preparation of Plasmid DNA by Small-scale Boiling Lysis Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks. - [Read Preparation of Plasmid DNA: Toothpick Minipreparation Protocol]

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Protocol describes how double-stranded plasmid DNA is denatured by alkali and annealed to an appropriate oligonucleotide primer, in preparation for dideoxy-sequencing reactions catalyzed by Sequenase. - [Read Preparing Denatured Templates for Sequencing by Dideoxy-mediated Chain Termination Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

In preparation for FLIM-FRET analysis, the appropriate donor and acceptor components must be introduced into live or fixed cells. The method of introduction depends on the nature of the components and the state of the cells. For example, plasmid DNAs encoding a protein of interest fused to a variant of GFP may be introduced into live cells by transfection or microinjection, whereas labeled antibodies are delivered by microinjection. - [Read Probing Protein Interactions Using GFP and FRET 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: Recombinant Protein Protocols

From plasmid to protein using bacterial expression. Transform appropriate DNA plasmid, Make a starter culture for protein expression, bacteria culture for protein expression. Sosnick Group Chicago. - [Read Protein Expression and Purification Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

A solution containing plasmid DNA, saturating amounts of ethidium bromide, and CsCl (44% w/v) is layered between two solutions of lesser (35% w/v CsCl) and greater density (59% w/v CsCl).  During centrifugation to equilibrium, the closed circular plasmid DNA and linear DNAs form bands at different densities. - [Read Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradient]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Solutions containing plasmid DNA are adjusted to a density of 1.55 g/ml with solid CsCl.  The intercalating dye, ethidium bromide, which binds differentially to closed circular and linear DNAs, is then added to a concentration of 200 mu;g/ml.  During centrifugation to equilibrium, the closed circular DNA and linear DNAs form bands at different densities. - [Read Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Protocol for purification of plasmid DNA by chromatography. - [Read Purification of Plasmid DNA by Chromatography Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Crude preparations of plasmid DNA are first treated with lithium chloride and Rnase (to remove RNA).  The plasmid DNA is then precipitated in a solution containing polyethylene glycol and MgCl2. - [Read Purification of Plasmid DNA by Precipitation with Polyethylene Glycol Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Protocol for quick DNA plasmid prep. Protocol gives very clean plasmid preps for restriction digests and cloning.  However, due to the alkaline lysis step, the DNA is often nicked and may not give exceptional sequence data. - [Read Quick DNA Plasmid Prep Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Protocol for quick DNA plasmid prep. This is a very fast mini-prep protocol which is suitable for sequence analysis and restriction digests. - [Read Quick DNA Plasmid Prep. Protocol]

Category: RNA Protocols: cDNA Libraries Library

Hoskins et al. 2005 NAR. "... describe a PCR-based method for directed screening of plasmid cDNA libraries. " - [Read Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP)]

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: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Contamination of plasmid DNA by fragments of DNA and RNA is reduced to an acceptable level by centrifugation through 1 M sodium chloride.  This method was devised by Brian Seed when he was a graduate student at Harvard University. - [Read Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

High-molecular-weight RNA and proteins can be precipitated from preparations of plasmid DNA by high concentrations of LiCl and removed by low-speed centrifugation. - [Read Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Li]

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: 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

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: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Protocol describing isolation of small scale plasmid from Xanthomonas. - [Read Small Scale Plasmid Isolation for Xanthomonas]

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

A vector that lacks inserts can significantly contaminate a DNA library. This contamination can occur either from self-ligation of single-cut vector or from uncut circular starting plasmid. - [Read Synthesis of DNA Libraries: Use of PCR to Analyze Ligation Substrates and Products]