BcaBEST RNA PCR Kit
BcaBEST RNA PCR Kit
Brand: Takara Bio.
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BcaBEST RNA PCR Kit
The BcaBEST RNA PCR Kit is designed to perform both reverse transcription and DNA amplification in a single tube. The kit uses BcaBEST DNA Polymerase, which contains both DNA polymerase and reverse transcriptase for first strand cDNA synthesis. In contrast to standard reverse transcriptases, BcaBEST Polymerase has a temperature optimum of 65°C, which permits cDNA synthesis from difficult and highly structured RNA templates. Bca-Optimized Taq Polymerase is used for second strand synthesis and subsequent PCR. This polymerase utilizes LA PCR technology for improved PCR length and accuracy. Random 9-mers, oligo-dT primers, or a specific downstream primer that acts as an antisense primer in PCR can be used for cDNA synthesis.
Overview
- Enables RT-PCR of long or difficult RNA templates
- Amplifies templates up to 5 kb in length
- Transcription at an optimum reaction temperature of 65°C
- Uses Bca-optimized Taq Polymerase, an LA technology-based enzyme that is optimized for use with BcaBEST DNA Polymerase
- Kit includes random 9-mers or oligo(dT) primers for first-strand cDNA synthesis. Specific downstream primers can be prepared by the user
- Procedure is performed in a single tube
- Reverse transcription is stopped via enzyme heat inactivation. PCR mixture is added directly to the tube
Applications
- Single-tube RT-PCR, using LA PCR technology for better PCR performance.
- Reverse transcription of RNA that is GC-rich or has high levels of secondary structure.
Amplification of human TRADD gene
Amplification of human TRADD gene. Amplification of the human TRADD (TNF receptor associated death domain) gene (494 bp, GC content 68.2%) was performed using total RNA from HeLa cells. Lanes 1 and 2 used the RNA PCR Kit, Ver. 2.1, and the BcaBest; RNA PCR Kit, Version 1.1, respectively. Lanes M contain a 100-bp ladder.
Components
| RR023A | |
| BcaBEST DNA Polymerase (22 U/µl) | 50 µl |
| RNase Inhibitor (40 U/µl) | 25 µl |
| Random 9-mers (50 µM) | 50 µl |
| Oligo dT Primer (50 µM) | 50 µl |
| RNase-Free dH2O (DEPC-treated) | 1 ml |
| Bca-Optimized Taq Polymerase (5 U/µl) | 25 µl |
| 2X Bca 1st Strand Buffer | 2 x 1.25 ml |
| 5X Bca 2nd Strand Buffer | 800 µl |
| dNTP Mixture (10 mM each) | 50 µl |
| MgSO4 (25 mM) | 500 µl |
| Control F-1 Primer (20 µmol/µl) | 25 µl |
| Control R-1 Primer (20 µmol/µl) | 25 µl |
| Positive Control RNA (1 µg/µl) | 25 µl |
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Amplification of human TRADD gene
Amplification of human TRADD gene. Amplification of the human TRADD (TNF receptor associated death domain) gene (494 bp, GC content 68.2%) was performed using total RNA from HeLa cells. Lanes 1 and 2 used the RNA PCR Kit, Ver. 2.1, and the BcaBest; RNA PCR Kit, Version 1.1, respectively. Lanes M contain a 100-bp ladder.
Components
| RR023A | |
| BcaBEST DNA Polymerase (22 U/µl) | 50 µl |
| RNase Inhibitor (40 U/µl) | 25 µl |
| Random 9-mers (50 µM) | 50 µl |
| Oligo dT Primer (50 µM) | 50 µl |
| RNase-Free dH2O (DEPC-treated) | 1 ml |
| Bca-Optimized Taq Polymerase (5 U/µl) | 25 µl |
| 2X Bca 1st Strand Buffer | 2 x 1.25 ml |
| 5X Bca 2nd Strand Buffer | 800 µl |
| dNTP Mixture (10 mM each) | 50 µl |
| MgSO4 (25 mM) | 500 µl |
| Control F-1 Primer (20 µmol/µl) | 25 µl |
| Control R-1 Primer (20 µmol/µl) | 25 µl |
| Positive Control RNA (1 µg/µl) | 25 µl |
