strand Protocols

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: 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: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

Protocol for apoptosis detection using TUNEL protocol. This technique uses the enzyme terminal deoxynucleotidyl transferase (TdT) to label cells that have oligonucleosomal nicks/strand breaks in their DNA. - [Read Apoptosis Detection: TUNEL Protocol]

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: 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: DNA Protocols: cDNA Library Protocols

Here, the DNA-RNA hybrids synthesized in Stage 1 are converted into full-length double-stranded cDNAs. The primers for synthesis of second-strand cDNA are created by RNase H, which introduces nicks into the RNA moiety of the cDNA-mRNA hybrids. E. coli DNA polymerase I extends the newly created 3'-hydroxyl termini, using the first-strand cDNA as a template. - [Read Construction of cDNA Libraries Protocol]

Category: Genetics and Genomics: Cancer Genetics Protocols: Single Strand Conformational Polymorphism(SSCP) Protocols

Single-strand conformational polymorphism (SSCP), one of several methods used to scan segments of DNA for mutations, exploits the electrophoretic differences in mobilities between single-stranded mutant and wild-type DNAs. - [Read Detection of Mutations by Single-strand Conformational Polymorphism Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis

First Strand cDNA Synthesis. Cebra-Thomas - [Read First Strand cDNA Synthesis]

Category: PCR Protocols: cDNA Synthesis Protocols

This cDNA synthesis system simplifies your work dramatically.  All reaction components are premixed and lyophylised.  You have to add your RNA and (for Your-Prime beads) the primer.  Another advantage of the system is a little number of pipetting steps required, and therefore reduced risk of Rnase contamination and RNA degradation. - [Read First strand cDNA synthesis with Ready-To-Go Beads Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

This method of first strand cDNA synthesis is more efficient than Ready-to-Go beads.  You can use as little as 100ng total RNA and still detect you gene expression in PCR. - [Read First strand cDNA synthesis with Super Script II Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis: RT-PCR and cDNA Synthesis FAQ

How much of the first strand reaction should I add to the PCR? Invitrogen - [Read How much of the first strand reaction should I add to the PCR?]

Category: RNA Protocols: cDNA Libraries Library

Library cDNA Synthesis. 1rst and 2nd strand cDNA synthesis. Dr. Simon Dawson - [Read Library cDNA Synthesis]

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

Protocol for mutation detection by single-strand conformational polymorphism (SSCP). Includes: PCR Protocol; SSCP Gels; - [Read Mutation Detection By Single-Strand Conformational Polymorphism (SSCP) Protocol]

Category: Genetics and Genomics: Cancer Genetics Protocols: Single Strand Conformational Polymorphism(SSCP) Protocols

Protocol for mutation detection by single-strand conformational polymorphism (SSCP). Includes: PCR Protocol; SSCP Gels. - [Read Mutation Detection By Single-Strand Conformational Polymorphism (SSCP) Protocol]

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: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this protocol, double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]

Category: PCR Protocols

Protocol describes how double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA.  RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue. - [Read Real-Time RT-PCR: cDNA Synthesis Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA.  RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue. - [Read Real-Time RT-PCR: cDNA Synthesis Protocol]

Category: DNA Protocols: DNA Sequencing Protocols

Protocol for single strand DNA prep for sequencing. Protocol works well from a 5 ml starting culture or a 1 ml starting culture. - [Read Single Strand DNA Prep for Sequencing Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols

Protocol for single strand DNA prep for sequencing. This protocol works well from a 5 ml starting culture or a 1 ml starting culture (adjust volume accordingly). - [Read Single Strand DNA Prep For Sequencing Protocol]