Description
General description
Rapid Amplification of cDNA ends (RACE) is a prevalent technique used to rapidly obtain full-length cDNA from partially known sequences. The 5′/3′ RACE kit contains Transcriptor Reverse Transcriptase and recombinant Terminal Transferase. Transcriptor Reverse Transcriptase transcribes full-length cDNA for the highly sensitive and rapid amplification of either 5′ or 3′ cDNA fragments up to 14 kb. Due to its thermostability (up to +65 °C), it can work with GC-rich templates with high secondary structure. High sensitivity can be achieved using Transcriptor Reverse Transcriptase, resulting in highly efficient cDNA synthesis and the generation of long RACE products. Recombinant Terminal transferase is used to add a homopolymeric A-tail to the 3′ end of the cDNA. The poly(A)⁺ tail decreases the likelihood of inappropriate truncation by the oligo(dT)-anchor primer, and overcomes the weaker A/T compared to the G/C hybridization. Moreover, longer stretches of A residues are required before the oligo(dT)-anchor primer can hybridize to an internal site and can truncate the amplification product. Tailed cDNA is amplified by PCR using a gene-specific primer and the oligo(dT)-anchor primer. The obtained cDNA is further amplified by a second PCR using a nested specific primer and the PCR-anchor primer, allowing RACE products to be cloned into an appropriate vector for subsequent studies.
Application
5′/3′ RACE Kit, 2nd Generation is suitable for
Structural and expression studies of RNA molecules
Generating full-length cDNAs
Isolation and characterization of 5′ or 3′ ends from low-copy RNA messages
first strand cDNA synthesis
Amplification and further cloning of rare mRNAs
Use in conjunction with exon-trapping methods
Products of the RACE reaction can be directly sequenced without cloning
Biochem/physiol Actions
Heat inactivation: Terminal Transferase: 70 °C for 10 minutes
Transcriptor Reverse Transcriptase: 85 °C for 5 minutes
Features and Benefits
Robust performance: Recombinant Transcriptor Reverse Transcriptase allows procession through regions of difficult secondary RNA structure.
Convenient: Function and expression studies of either 5′ or 3′ end of the RNA can be performed with the same kit.
Reliable: dA tailing of cDNA with Recombinant Terminal Transferase decreases the likelihood of inappropriate truncation.
Reproducible: Oligo dT-anchor primer with non 3′dT ensures correct binding to the inner end of the poly (A) tail.
Produce long fragments: Generate cDNA up to 14kb in length with Transcriptor Reverse Transcriptase.
The control reaction includes transcription of the control RNA into first-strand cDNA using the control primer neo1/rev. Amplification of the cDNA is performed using the control primer neo2/rev and neo3/for to obtain a 157-bp PCR product. Tailing of the purified cDNA is performed with dATP. Amplification of the tailed cDNA is performed with oligo dT-anchor primer and the control primer neo2/rev to obtain a 293-bp PCR product.
Packaging
1 kit containing 12 components
Other Notes
For life science research only. Not for use in diagnostic procedures.
