oligonucleotides Protocols

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: Transfection Protocols: Stable Transfection Protocols

The AfCS is utilizing antisense technology to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. This can be achieved by the transfection of gene-specific antisense oligonucleotides (ASOs). The following procedure involves the transfection of ASOs into RAW 264.7 cells using FuGENE 6 transfection reagent. Subsequently, the isolated total RNA or protein from these transfected cells can be used to assess the level of mRNA or protein knockdown, respectively. - [Read Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish]

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: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

This protocol describes how to use DIG Chem-Link to directly label any DNA [e.g. plasmids, PCR products, cDNA prepared from mRNA] or RNA (e.g. total RNA, poly(A)+ mMRNA). The DIG Chem-Link or Biotin Chem-Link may also be used to label oligonucleotides. Includes: Required Purity of DIG Chem-Link Templates; Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Key Product Required for Direct Labeling of DNA or RNA; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

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: PCR Protocols

Method uses PCR to amplify and display many cDNAs derived from the mRNAs of a given cell or tissue type.  The method relies on two different types of synthetic oligonucleotides: anchored antisense primers and arbitrary sense primers.  A typical anchored primer is complementary to approx. 13 nucleotides of the poly(A) tail of mRNA and the adjacent two nucleotides of the transcribed sequence. - [Read Differential Display-PCR Protocol]

Category: DNA Protocols: DNA Cloning

Agarose gel purification, Annealing and extending, oligonucleotides, Ethanol Precipitation, Ligation Miniprep, Oligonucleotide purification, Recovering DNA bands, Restriction digest Gene Clean. The Hu Lab. - [Read DNA cloning Procedures]

Category: DNA Protocols: EMSA DNA-Protein Protocols

EMSA probe creation using ds Oligonucleotides by annealing two complementary oligos, and Klenow for probe creation. - [Read EMSA using ds Oligonucleotides]

Category: DNA Protein Interactions Protocols: Electrophoretic Mobility Shift Assays EMSA Protocols

Protocol for EMSA using ds oligonucleotides. - [Read EMSA using ds Oligonucleotides Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Labeling Protocols

Hybridization is carried out in conventional aqueous solvents at a temperature well below the predicted melting temperature.  Nonspecific hybrids are then removed by washing at high stringency in buffers containing quaternary salts.  Tetramethylammonium chloride (TMACl) is used with probes that are 14-50 nucleotides in length, whereas tetraethylammonium chloride (TEACl) is used with longer oligonucleotides. - [Read Hybridization of Oligonucleotide Probes in Aqueous Solutions Protocol]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol for the identification of single bacterial cells using DIG-labeled oligonucleotides. Includes: Organisms and growth conditions; Cell fixation and preparation of cell smears; DIG labeling of oligonucleotides with DIG-ddUTP; In situ hybridization using digoxigenin-labeled oligonucleotides; Detection of DIG-labeled oligonucleotides with fluorescently labeled anti-DIG Fab fragments; Detection of DIG-labeled oligonucleotide.
 - [Read Identification of Single Bacterial Cells using DIG-Labeled Oligonucleotides Protocol]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for identification of single bacterial cells using DIG-labeled oligonucleotides. Includes: Organisms and growth conditions; Cell fixation and preparation of cell smears; DIG labeling of oligonucleotides with DIG-ddUTP; In situ hybridization using digoxigenin-labeled oligonucleotides. - [Read Identification of Single Bacterial Cells Using DIG-Labeled Oligonucleotides Protocol]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for in vitro mutagenesis using double-stranded DNA templates. Two oligonucleotides are used to prime DNA synthesis catalyzed by a high-fidelity thermostable polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and occupy the same starting and ending positions on opposite strands of the plasmid DNA. - [Read In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with DpnI]

Category: DNA Microarray Protocols

Making a scanning array: in situ synthesis of oligonucleotides on a solid substrate. Elder et al., Oxford Practical Approach Series. - [Read Making a scanning array: in situ synthesis of oligonucleotides on a solid substrate]

Category: Microarray Protocols: Amino-allyl Microarray Methods

Protocol details the preparation of fluorescently labeled target samples (aminoallyl method) and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides. The procedure requires a minimum of 3 µg of purified total RNA as starting material. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized]

Category: Microarray Protocols: Microarray DNA Labeling Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides. The procedure requires a minimum of 3 µg of purified total RNA as starting material. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized Oligo Array]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides.  The procedure requires a minimum of 3 µg of purified total RNA as starting material. Includes: cDNA Synthesis; Fluorescent cRNA Synthesis; cRNA Precipitation and Cleanup; cRNA Quantification; Hybridization; Washing. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized Oligo Arrays]

Category: PCR Protocols: cDNA Synthesis Protocols

In MOPAC, the amino-terminal and carboxy-terminal sequences of a peptide are used to design two redundant families of oligonucleotides encoding the aminoand carboxy-terminal sequences of the peptide.  The primers are used either to amplify a segment of cDNA prepared by RT-PCR from a tissue known to express the protein or to amplify a segment of DNA from an established genomic or cDNA library. - [Read Mixed Oligonucleotide-primed Amplification of cDNA MOPAC Protocol]

Category: Real Time PCR: Real Time PCR Information

Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. Livak et al., 1995. - [Read Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for d]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for phosphorylating the 5' termini of oligonucleotides. - [Read Phosphorylating the 5' Termini of Oligonucleotides Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a method for observing and measuring the movement of RNA molecules in the nucleus of living mammalian cells. Caged fluorescein-labeled DNA oligonucleotides are introduced into living mammalian cells, where they demonstrably hybridize to complementary RNA. After site-specific photoactivation at desired sites within the cell, the RNA movements away from those sites are followed and digitally recorded using a rapid acquisition microscopy system. - [Read Photoactivation-Based Labeling and In Vivo Tracking of RNA Molecules in the Nucleus]

Category: Chromatography Protocols

This protocol describes the preparation of concatamerized oligonucleotides and their coupling to cyanogen-bromide-activated Sepharose. The procedure uses a commercially activated resin, which can be purchased as a lyophilized powder. Keith Brocklehurst et al. - [Read Preparation of DNA Affinity Resin - Subscription Required]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Procedures for labeling DNA, RNA, and oligonucleotides with DIG, biotin, or fluorochromes. Includes: Random primed labeling of ds DNA with DIG-, Biotin- or Fluorescein-High Prime reaction mix; PCR labeling of ds DNA with the PCR DIG Probe Synthesis Kit or PCR Labeling Mixes. - [Read Procedures for Labeling DNA, RNA, and Oligonucleotides with DIG, Biotin, or Fluorochromes]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Procedures for labeling DNA, RNA, and oligonucleotides with DIG, Biotin, or Fluorochromes. Includes: Random primed labeling of ds DNA with DIG-, Biotinor Fluorescein-High Prime reaction mix; PCR labeling of ds DNA with the PCR DIG Probe Synthesis Kit or PCR Labeling Mixes. - [Read Procedures for Labeling DNA, RNA, and Oligonucleotides with DIG, Biotin, or Fluorochromes]

Category: Nucleic Acid Purification Protocols

Radiolabeled nucleic acids, including oligonucleotides, can be separated from unincorporated radiolabel by quantitative, differential precipitation with the cationic detergent cetylpyridinium bromide (CPB).  The nucleic acids are first precipitated from aqueous solution with CPB. - [Read Purification of Radiolabeled Oligonucleotides by Precipitation with Cetylpyridinium Bromide Protocol]