oligonucleotide Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "3'-end" partial cDNA clones, mRNA is reverse-transcribed using a "hybrid" primer (Qtotal, QT) that consists of two mixed bases (GATC/GAC followed by [T]17) and a unique 35-base oligonucleotide sequence (QI-QO).  Amplification is then performed using a primer containing part of this sequence (Qouter, Qo) (which now binds to each cDNA at its 3'-end) and a primer derived from the gene of interest, GSP1 (gene-specific primer 1). - [Read 3'-End cDNA Amplification Using Classic RACE 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: 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: Cell Biology and Cell Culture: Apoptosis Protocols: NF-kB EMSA Protocols

Detailed NF-kB EMSA Protocol. Includes Nuclear extract preparation, Labeling of the oligonucleotide probe: T4 Polynucleotide Kinase Reaction, Supershift assay to identify NF-kB, and recipes for EMSA buffers. Celldeath.de - [Read Detailed NF-kB EMSA Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes. - [Read Detection of mRNA in Tissue Sections Using DIG-labeled RNA and Oligonucleotide Probes 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: E Coli Protocols: E coli Nucleic Acid Protocols

E.coli total RNA labeling protocol for high density oligonucleotide array. Includes: RNA Preparation; Digest RNA and Purification of cDNA; Purify cDNA with Qiaquick PCR purification kit; cDNA Fragmentation and end labeling; Labeling with Terminal Transferase. - [Read E.coli Total RNA Labeling Protocol for High Density Oligonucleotide Array]

Category: Microarray Protocols: Microarray DNA Labeling Protocols

Protocol for e.coli total RNA labeling for high density oligonucleotide array. - [Read E.coli Total RNA Labeling Protocol for High Density Oligonucleotide Array]

Category: PCR Protocols: PCR Optimization

Method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.  The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. - [Read Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Method assesses cellular mRNA transcripts in tissue sections and cell cultures using unique short anti-sense primers directed against sequences in particular protein(s). The unlabeled synthetic cDNA oligonucleotide primers are extended complementary to a sense mRNA transcript using reverse transcriptase and labeled through incorporation of a fluorescent-labeled dUTP nucleotide base. This procedure provides rapid detection of low abundance mRNA messages that can be related to other cellular.... - [Read Fluorescent In Situ Transcription in Cells and Tissues 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: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of biotin-labeled target samples and hybridization of these samples to an Affymetrix in situ synthesized oligonucleotide GeneChip array.  The procedure requires a minimum of 5 µg of purified total RNA as starting material. - [Read Microarray Protocol for Affymetrix In Situ Synthesized Oligo Arrays]

Category: Mass Spectrometry Protocols

Millipore's ZipTip Protocol. Intended for purifying and concentrating femtomoles to picomoles of protein, peptide or oligonucleotide samples prior to analysis, providing better data quality. Millipore. - [Read Millipore's ZipTip Protocol]

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: Cytogenetics Protocols: In Situ Hybridization Protocols

Discusses the effects of various components of the hybridization solution on the rate of renaturation and thermal stability of DNA hybrids free in solution. Includes: The main parameters that influence hybridization; Additional hybridization variables; Competition in situ hybridization; Oligonucleotide hybridization; Standard in situ hybridization conditions. - [Read Nucleic Acid Hybridization General Aspects]

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

Protocol for oligonucleotide 3’-end labeling with DIG-ddUTP or Biotin-ddUTP. - [Read Oligonucleotide 3’-End Labeling with DIG-ddUTP or Biotin-ddUTP
Protocol  ]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol for oligonucleotide 3’-end labeling with DIG-ddUTP or Biotin-ddUTP.
- [Read Oligonucleotide 3’-end Labeling with DIG-ddUTP or Biotin-ddUTP Protocol]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

Information for oligonucleotide details required for PCR. Includes: Primer choice; UPTAG PRIMER; DNTAG PRIMER; UP_45 and DOWN_45 PRIMERS; UP_90 and DOWN_90 PRIMERS. - [Read Oligonucleotide Details for PCR]

Category: Nucleic Acid Purification Protocols

Protocol for oligonucleotide purification. - [Read Oligonucleotide Purification Protocol]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol for oligonucleotide tailing with a DIG-dUTP, Biotin-dUTP, or Fluorescein-dUTP mixture.
- [Read Oligonucleotide Tailing with a DIG-dUTP, Biotin-dUTP, or Fluorescein-dUTP Mixture Protocol]

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

Protocol for oligonucleotide tailing with a DIG-dUTP, Biotin-dUTP, or Fluorescein-dUTP mixture. - [Read Oligonucleotide Tailing With a DIG-dUTP, Biotin-dUTP, or Fluorescein-dUTP Mixture Protocol]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for oligonucleotide-directed mutagenesis by elimination of a unique restriction site (USE mutagenesis). - [Read Oligonucleotide-directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis)]

Category: Cloning Protocols: Mutagenesis Protocols

Protocool for oligonucleotide-directed mutagenesis of single-stranded DNA. - [Read Oligonucleotide-directed Mutagenesis of Single-stranded DNA Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Protocol for PCR genotyping from tail DNA. This protocol works well for a variety of genes and primer pairs including Tg and KO alleles. Oligonucleotide melting temperatures between 60° and 65° seem to work well. - [Read PCR Genotyping from Tail DNA Protocol]