race 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: 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: PCR Protocols: cDNA Synthesis Protocols

3'-RACE reactions are used to isolate unknown 3' sequences or to map the 3' termini of mRNAs onto a gene sequence.  3'-RACE requires knowledge of a small region of sequence within either the target RNA or a partial clone of cDNA.  A population of mRNAs is transcribed into cDNA with an adaptor-primer consisting at its 3' end of a poly(T) tract and at its 5' end of an arbitrary sequence of 30-40 nucleotides. - [Read Rapid Amplification of 3' cDNA Ends 3'-RACE Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs.  The technique requires knowledge of a small region of sequence within the partial cDNA clone.  During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence. - [Read Rapid Amplification of 5' cDNA Ends 5'-RACE Protocol]