cdna 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: RNA Protocols: 5' RACE - Rapid Amplification of cDNA Ends Protocols

Can also use 96-well plates. First Strand cDNA Synthesis, First Size selection, second 2nd strand cDNA synthesis. Bay Area Functional Genomics Consortium, UCSF. - [Read 5' RACE Protocol For Generation of Seq. Tags]

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: Bioinformatics

Using AFPredictor, it was demonstrated that ‘ordered surface carbons’ (OSCs) are a distinguishing feature of AFPs and, more specifically, their ice-binding surfaces. AFPredictor identified AFPs from within a large set of structures with greater than 99% specificity. Furthermore, it was used to identify a novel ice-binding protein by screening a library of homology modeled structures based on cDNA sequences obtained from cold-acclimated winter rye (Secale cereale). - [Read A Computational Screening protocol for Antifreeze/Ice-Structuring Proteins]

Category: DNA Microarray Protocols

Micro Array Fabrication, Probe Preparation and Hybridization, and Data Collection, Normalization and Analysis. Hegde, et al. biotechniques 2000. - [Read A Concise Guide to cDNA Microarray Analysis – II PDF.]

Category: Signalling Signal Transduction Protocols

Protocol for Akt/PKB kinase assay. Protocol includes: Akt lysis buffer; Kinase Buffer; 1.5x Akt kinase reaction buffer; Start solution; Lipofectamine PLUS; CDNA synthesis (Roche #1483?88). - [Read Akt/PKB Kinase Assay]

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: DNA Microarray Protocols

This protocol describes the labeling of eukaryotic RNA with aminoallyl labeled nucleotides via first strand cDNA synthesis followed by a coupling of the aminoallyl groups to either Cyanine 3 or 5 (Cy 3/Cy5) fluorescent molecules. Hasseman. TIGR Microarr - [Read Amino-allyl Labeling]

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: DNA Protocols: cDNA Library Protocols

This stage achieves four goals: polishing the ends of double-stranded DNA, ligation of synthetic linkers or adaptors, digestion of the attached linkers to create cohesive termini, and preparing the cDNA for cloning. - [Read Attachment of Linkers or Adaptors for Construction of cDNA Libraries]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

The ability to synthesize RNA in the lab is critical to many techniques.Radiolabeled and nonisotopically labeled RNA probes, generated in small scale transcription reactions can be used in blot hybridizations and nuclease protection assays. This article includes information on: Requirements For Transcription, RNA Phage Polymerases, Template Options: Plasmids, PCR Products, Oligonuclotides and cDNA, Sense or Antisense, Conventional Or Large Scale Synthesis, Products for In Vitro Transcription. - [Read Basic Information on In Vitro Transcription]

Category: RNA Protocols: cDNA Libraries Library

Includes procedures for cDNA screens using secondary and tertiary screening. Dr. Bart Frank Lab. - [Read Benton Davis Blots for cDNA Libraries]

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: Genetics and Genomics: Cancer Genetics Protocols

CDNA/AFLP. A tool for transcriptome analysis. - [Read cDNA / AFLP]

Category: RNA Protocols: RT-PCR and cDNA synthesis

Method of synthesizing cDNA from Frozen Tissue Sections Protocol. Includes LCM - Laser Capture Microdissection Protocols. NIH - [Read cDNA from Frozen Tissue Sections and LCM]

Category: RNA Protocols: cDNA Libraries Library

cDNA Library Construction Lecture. Dr.Blaber Lab - [Read cDNA Library Construction Lecture]

Category: RNA Protocols: cDNA Libraries Library

cDNA Library Screening Protocol - ZAPII Detailed Protocol. XLI-Blue MRF' and cDNA phage library screening. Dr. Laurie. University of Virginia Health Science Center - [Read cDNA Library Screening Protocol - ZAPII]

Category: RNA Protocols: cDNA Libraries Library

Protocol for cDNA synthesis and cloning cDNA into plasmid vector. 1st Strand cDNA Synthesis, and determine the efficiency of first strand cDNA synthesis. Also includes second Strand cDNA Synthesis. Dr.Frank - [Read cDNA Synthesis and Cloning]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol describes the real-time PCR using RNA purified from laser-captured tissues Laser Capture Microdissection (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cells. - [Read cDNA Synthesis and Real-Time PCR Using RNA from Laser-Captured Cells Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis

cDNA Synthesis from MOLT-4 Cells. First strand of cDNA synthsis. Cungui Mao - [Read cDNA Synthesis from MOLT-4 Cells]

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: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for Chem-Link labeling of DNA or RNA with DIG or Biotin. Includes: Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]