reverse Protocols

Category: RNA Protocols: 3' RACE Rapid Amplification of cDNA Ends Protocols

PCR cycle steps, protocol using SuperScript II reverse transcriptase (Life Technologies) and Taq polymerase, Gene specific primer, T17 Adapter primer and an Adapter primer. Dr.Dawson, Nottingham. - [Read 3' Rapid Amplification of cDNA Ends (RACE) Protocol]

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: PCR Protocols: cDNA Synthesis Protocols

To generate "5'-end" partial cDNA clones using classic RACE, the first-strand products are generated by reverse transcription (primer extension) from a known gene-specific primer (GSP-RT).  Then, a poly(A) tail is appended using terminal deoxynucleotidyltransferase (Tdt) and dATP.  Amplification is carried out using three primers. - [Read 5'-End cDNA Amplification Using Classic RACE Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

New RACE, a variation of RNA ligase-mediated-RACE (RLM-RACE) (Liu and Gorovsky 1993) departs from classic RACE (see 5'-End cDNA Amplification Using Classic RACE) in that an "anchor" primer is attached to the 5'-end of the mRNA before the reverse transcription step; hence the anchor sequence becomes incorporated into the first-strand cDNA if, and only if, the reverse transcription proceeds through the entire length of the mRNA of interest. - [Read 5'-End cDNA Amplification Using New RACE Protocol]

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

Alkaline agarose gels are used chiefly to measure the size of first and second strands of cDNA (Construction of cDNA Libraries Stage 1: Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase) and to analyze the size of the DNA strand after digestion of DNA-RNA hybrids with nucleases such as S1. - [Read Alkaline Agarose Gel Electrophoresis Protocol]

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: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses a single thermostable RNA polymerase to perform high-specificity RT-PCR.  A high-temperature RT reaction is followed by PCR amplification of the cDNA using a single thermostable poymerase, the GeneAmp AccRT RNA PCR enzyme from Applied Biosystems.  The high temperature of the RT reaction enhances the specificity of primer binding and also reduces secondary structure in the template, thereby increasing the efficiency of polymerization. - [Read Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium]

Category: Genetics and Genomics: Cancer Genetics Protocols

Anatomy of a comparative gene expression study. Includes: Choosing Cell Populations; mRNA Extraction and Reverse Transcription; Fluorescent Labeling of cDNA's; Hybridization to a DNA Microarray; Scanning the Hybridized Array; Interpreting the Scanned Image. - [Read Anatomy of a Comparative Gene Expression Study]

Category: Microarray Protocols: Microarray Analysis Protocols

Information on anatomy of a comparative gene expression study.  Includes: Choosing cell populations; mRNA Extraction and Reverse Transcription; Fluorescent labeling of cDNA's; Hybridization of DNA microarray; Scanning the hybridized array; Interpreting the scanned image. - [Read Anatomy of a Comparative Gene Expression Study]

Category: Cell Biology and Cell Culture

Protocol for automated imaging-based high-throughput screening for small molecules that reverse cellular senescence. - [Read Automated Imaging-Based High-Throughput Screening: Small Molecules that Reverse Cellular Senescence]

Category: PCR Protocols: cDNA Synthesis Protocols

This method, for the selective amplification of full-length cDNA ends, involves the addition of an adapter during reverse transcription.  This method takes advantage of the propensity of Moloney murine leukemia virus reverse transcriptase (MMLV RT) to append two to four cytosines to the 3'-end of newly synthesized cDNA strands.  The additional residues are added when the enzyme reaches the 5'-cap structure at the end of the mRNA template. - [Read Cap-Switching RACE Protocol]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

SPHEROPLAST CELLS, WASH SPHEROPLASTS, SONICATE SPHEROPLASTS, CHROMATIN SOLUTION, SET UP IMMUNOPRECIPITATIONS, REVERSE FORMALDEHYDE CROSSLINKS. Paul - Koshland lab. - [Read CHROMATIN IMMUNOPRECIPITATION (CHIP) PROTOCOL FOR YEAST]

Category: Epigenetics and Methylation Protocols: ChIP Chromatin Immunoprecipitation Protocols

Chromatin immunoprecipitation protocol to analyze histone modifications in Arabidopsis thaliana. Werner Aufsatz, Matzke Lab. Gregor Mendel Institute of Molecular Plant Biology, Austria. Includes Chromatin Crosslinking, Chromatin preparation, Pre-clearing and immuno precipitation (IP), Collection,washes and elution of immune complexes, Reverse crosslinking and DNA cleanup - [Read Chromatin immunoprecipitation protocol to analyze histone modifications in Arabidopsis thaliana]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In this protocol, sample and competitor RNAs are reverse transcribed (separately) in a pilot experiment.  A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. Procedure provides an approximate copy number for the sample, which is then fine-tuned by repeating the experiment with a series of twofold dilutions of competitor.  The experiment includes controls for sample-to-sample variations in RT efficiency. - [Read Competitive RT-PCR: Estimation of Copy Number Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor.  This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest.  Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out. - [Read Competitive RT-PCR: Preparation of Competitor RNA 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: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation of mRNA-protein complexes. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. - [Read Immunoprecipitation of mRNA-Protein Complexes Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Retroelements and their derivatives are a ubiquitous and abundant component of plant genomes. Major classes of retroelements include the Pseudoviridae (Ty1-copia ), the Metaviridae (Ty3 -gypsy) and the Retroposineae LINE (non-LTR) groups. All reverse transcribing elements can be included in a universal classification. Includes: Pseudoviridae (Ty1-copia) Degenerate Primers; Metaviridae (Ty3-gypsy) Element Degenerate Primers; LINE Element Degenerate Primers; PCR Programmes. - [Read Isolation of Retroelement from Plant Genomic DNA]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures. - [Read Older RT-PCR and PCR Techniques Page.]

Category: PCR Protocols

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures. - [Read Older RT-PCR and PCR Techniques Page.]

Category: RNA Protocols: RT-PCR and cDNA synthesis

Protocol for First-strand cDNA Synthesis (PCR template generation with M-MuLV Reverse Transcriptase) - [Read Protocol for First-strand cDNA Synthesis Fermentas]

Category: DNA Microarray Protocols

Reverse transcriptase labeling with Cy3/Cy5, Staining of total DNA on slides, construction, Laser scanning, Spot quantitation, Statistics, sample tracking. Vodkin Laboratory. U.Illinois. - [Read Protocols for microarray development in use in the Vodkin Laboratory]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Standard PCR Protocol

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures. Informative Guide. Jack Vanden Heuvel. - [Read Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures]

Category: PCR Protocols: Standard PCR Protocol

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures. Informative Guide. Jack Vanden Heuvel. - [Read Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

Protocol is the second in a set of three, describing fluorescent mRNA differential display (FDD or FDDRT-PCR).  For the purposes of FDD gene expression analysis, as well as any other RNA-based gene expression technologies, contaminating genomic DNA must be removed before reverse transcription and subsequent PCR. - [Read Removal of Genomic DNA from Total RNA for Use in Fluorescent mRNA Differential Display Protocol]