target Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

An oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR.  Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs - [Read Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol]

Category: RNAi Technology Protocols

Protocol describes the preparation of an shRNA-expressing Gateway entry vector.  This process is preceded by the design and synthesis of target gene-specific sense and antisense oligonucleotides which, when annealed, will constitute an shRNA cassette. - [Read Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for bidirectional, blunt-end cloning of DNA fragments.  The target DNA is PCR amplified and 3'-extensions are polished with Pfu DNA polymerase.  The amplicon is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent Escherichia coli.  A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Bidirectional Cloning of PCR Products Protocol]

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo. - [Read Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Calibration Protocols

Flow cytometers must be calibrated prior to fluorescence intensity measurements because of inherent instrument variability. To correct for this variability, a standard particle (fixed chicken red blood cells, or CRBCs) must be analyzed on the instrument prior to each experiment and photomultiplier tube (PMT) voltages adjusted accordingly to place the CRBC fluorescence emission peaks into predetermined target channels. - [Read Calibration of Becton Dickinson Flow Cytometers for Relative Fluorescence Intensity Measurements]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Choosing the right labeling method for your hybridization experiment. Includes: Homogeneous labeling methods for DNA; Homogeneous labeling methods for RNA; Stability of probe-target interaction; Nonradioactive labeling of oligonucleotides; Double-stranded versus single-stranded probes. - [Read Choosing the Right Labeling Method for your Hybridization Experiment]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol for cloning of amplified miRNA target sites into 3'-UTR of luciferase reporter vector. - [Read Cloning of Amplified miRNA Target Sites into 3'-UTR of Luciferase Reporter Vector 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: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol describes the use of western blotting to analyze the RNAi-induced depletion of target protein from mammalian cells.  Immunofluorescence can be used as an alternative. - [Read Detection of RNAi-Induced Protein Knockdown in Mammalian Cells by Western Blotting Protocol]

Category: Western Blot Protocols

Protocol describes the use of western blotting to analyze the RNAi-induced depletion of target protein from mammalian cells.  Immunofluorescence can be used as an alternative. - [Read Detection of RNAi-Induced Protein Knockdown in Mammalian Cells by Western Blotting Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for directional blunt-end cloning of DNA fragments.  The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA.  The products of the ligation reaction are used to transform competent Escherichia coli.  A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Directional Cloning of PCR Products Protocol]

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

DNA Affinity Chromatography, DNA affinity chromatography can be a low-tech method using gravity flow at 4°C, a disposable chromatography column, and DNA affinity resin prepared in the laboratory (see Preparation of a DNA Affinity Column). Include 10-20% glycerol and 0.025-0.1% NP-40 in the column buffers to suppress losses due to nonspecific adsorption of protein to surfaces. Load the protein in a buffer that is compatible with binding of the protein to its target site. Keith Brocklehurst et al - [Read DNA Affinity Chromatography Using Gravity Flow - Subscription Required]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the expression of cloned genes in E. coli using IPTG-inducible promoters. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying IPTG-inducible promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using IPTG-inducible Promoters Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage {lambda} pL promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for expression of cloned genes in E. coli using the bacteriophage lambda pL promoter. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage lambda pL promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the expression of cloned genes in E. coli using the bacteriophage T7 promoter. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage T7 promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Acidocalcisomes, the dense acidic calcium-storing organelles, which were originally identified in Trypanosoma cruzi, have no parallels in mammalian cells. They thus represent a unique functional characteristic, not shared by the host and hence offer an important potential target for chemotherapy of Chagas disease. - [Read Fractionation of Acidocalcisomes and Other Organelles from Trypanosoma, Leishmania, Chlamydomonas]

Category: Antibody Protocols: Antibody Production Protocols

Novel strategy of immunizing a phosphorylated peptide or a synthetic phosphopeptide, which corresponds to the protein phosphorylated at a targeted residue. Method has been applied to the production of antibodies that can specifically recognize the other types of site-specific protein modification, such as acetylation, methylation, and proteolysis. - [Read Functional Analyses for Site-Specific Phosphorylation of a Target Protein in Cells]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

To determine the selectivity of the inhibitors identified during screening efforts we developed gel-elongation assay using crude bacterial lysate directly to determine the target specificities of fatty acid synthesis inhibitors. - [Read Gel Elongation Assay for Type II Fatty Acid Synthesis Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Polyacrylamide Gels Protocols

Protocol exploits differences in electrophoretic mobility through a nondenaturing polyacrylamide gel between a rapidly migrating target DNA and a more slowly migrating DNA-protein complex. - [Read Gel Retardation Assays for DNA-binding Proteins Protocol]

Category: Lipid Protocols

New screening efforts and chemical modifications of existing compounds have been attempted to identify more selective and potent inhibitors. To determine the selectivity of the inhibitors identified during screening efforts we developed gel-elongation assay using crude bacterial lysate directly to determine the target specificities of fatty acid synthesis inhibitors. - [Read Gel-elongation Assay for Type II Fatty Acid Synthesis Protocol]

Category: Gene Delivery Protocols: Biolistics Protocols

The technique has many advantages—plasmids may be used for delivery, DNA theoretically can be delivered to any cell type, and genes may be delivered to cells in vitro, ex vivo, or in vivo. DNA-coated gold particles are distributed evenly along the length of the tubing, which is subsequently cut into short sections of cartridges to be used in a gene gun. The Helios Gene Gun uses a pulse of helium to launch the DNA-coated particles, spreading them onto the target cells. - [Read Gene Delivery to Skin Using Biolistics Protocol]

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

Shotgun sequencing of a large segment of DNA involves random fragmentation of the target region into smaller segments that are subsequently cloned into a bacteriophage M13 vector. The goal is to create a library of overlapping clones that provide at least fivefold coverage over the entire length of the target fragment. - [Read Generation of a Library of Randomly Overlapping DNA Inserts Protocol]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for the generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. - [Read Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol]

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

The double-stranded DNA of recombinant plasmid, phagemid, or bacteriophage M13 replicative form DNA is digested with two restriction enzymes whose sites of cleavage both lie between one end of the target DNA and the binding site for universal primer. The enzyme that cleaves nearer the target sequence must generate either a blunt end or a recessed 3' terminus; the other enzyme must generate a four-nucleotide protruding 3' terminus. - [Read Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol]