linear Protocols

Category: Protein Protocols: Protein Quantitation Concentration Protocols

Absorbance assays are fast and convenient, since no additional reagents or incubations are required.  No protein standard need be prepared.  The assay does not consume the protein.  The relationship of absorbance to protein concentration is linear.  Because different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures. - [Read Absorbance Assay 280 nm]

Category: Cloning Protocols

Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms. - [Read Attaching Adaptors to Protruding Termini Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Protocol for blunt-end cloning of PCR products. Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids.  The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves.  I - [Read Blunt-end Cloning of PCR Products Protocol]

Category: C. elegans Protocols: C. elegans Genetics Protocols

Protocol for C. elegans RNAi. Includes: Transformation of competent cells; Blunt-end ligation; Preparation of competent cells; Dephosphorylation of linear plasmid DNA; Restriction Digest: EcoRV; Insert amplification from gDNA; Gel purification: QiaQuick gel purification kit; Mini-prep; Transformant Screening; Bacterial preparation and induction; Preparation of worms for RNAi feeding. - [Read C. elegans RNAi Protocol]

Category: Molecular Biology Protocols: Carbohydrate Protocols

Characterization of Protein Glycosylation Using Chip-Based Nanoelectrospray with Precursor Ion Scanning Quadrupole Linear Ion Trap Mass Spectrometry. Sheng Zhang1 and Brian L. Williamson. Journal of Biomolecular Techniques, 16:209-219 2005. - [Read Characterization of Protein Glycosylation Using Chip-Based Nanoelectrospray with Precursor Ion Scann]

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: Antibody Protocols: Epitope Mapping Antibody Protocols

The simplest way to determine whether two monoclonal antibodies bind to distinct sites on a protein antigen is to carry out a competition assay. The assay can be used with antibodies that bind both conformational and linear epitopes, and it is most useful in the analysis of monoclonal antibody specificity because polyclonal sera typically recognize multiple different epitopes. - [Read Epitope Mapping by Competition Assay Protocol]

Category: Antibody Protocols: Epitope Mapping Antibody Protocols

A set of overlapping synthetic peptides is synthesized, each corresponding to a small segment of the linear sequence of a protein antigen and arrayed on a solid phase. The panel of solid-phase peptides is then probed with a test antibody, and bound antibody is detected using an enzyme-labeled secondary antibody. This method is very rapid and can be extraordinarily successful. - [Read Epitope Mapping Using Synthetic Biotin-Labeled Peptides Protocol]

Category: Cell Biology and Cell Culture

Cell fractionation of cellular components using Percoll a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. Percoll becomes a simple matter to establish a linear density gradient. Organelle separations are much easier to accomplish on Percoll density gradients than on sucrose gradients. - [Read Equilibrium Density Gradient Percoll Protocol]

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol describes methods for isolation of DNA from a strain of S. cerevisiae carrying a recombinant YAC. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. The method is suitable for preparing DNA that will be used for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that do not require intact high-molecular-weight DNA. - [Read Growth of S. cerevisiae and Preparation of DNA Protocol]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to measure and monitor linear growth rate. - [Read How to Measure and Monitor Linear Growth Rate]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to obtain unbroken linear asci that are extruded individually from the perithecium. - [Read How to Obtain Unbroken Linear Asci that are Extruded Individually From the Perithecium]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol fopr markers of pulsed-field gel electrophoresis. Markers for pulsed-field gel electrophorsis can be generated by ligation of linear monomers of bacteriophage {lambda} DNA (48.5 kb) into a nested series of concatemers.  This procedure yields a series of concatemers that contain up to 20 tandemly arranged copies of bacteriophage DNA. - [Read Markers for Pulsed-field Gel Electrophoresis Protocol]

Category: Pharmacology and Toxicology Protocols: Pharmacokinetics Protocols

Non-linear Regression Programs for the Analysis of Pharmacokinetic and Pharmacodynamic Data - [Read Non-linear Regression Programs for the Analysis of Pharmacokinetic and Pharmacodynamic Data]

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: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In the first part of this protocol, the linear range of amplification is determined by carrying out 10 identical PCRs in the presence of [{alpha}-32P]dCTP and stopping one reaction after every two cycles.  Amplification products are quantified on a denaturing polyacrylamide gel and the results plotted on a graph (counts per minute vs. cycle number).  Total RNA is used as an internal control. - [Read Relative RT-PCR: Determining the Linear Range of Amplification and Optimizing the Primers:Competimer]

Category: Molecular Biology Protocols: DNA Ligation Protocols

Self-circularization of Linear DNA (with T4 DNA Ligase). Fermentas - [Read Self-circularization of Linear DNA (with T4 DNA Ligase)]