2X RT-PCR Master Mix is used for real-time qualitative and quantitative multiplex RT-PCR amplifications with TaqMan probes.
The kit is designed for multiplex RT-PCR with up to five pairs of primers and five TaqMan probes.
The master mix is a premixed, 2X concentrated solution that has all the components except for gene-specific primers, probes and RNA templates.
In a single-tube format, multiplex RNA templates are first converted to cDNAs by a reverse transcriptase, then a DNA-dependent DNA polymerase amplifies the cDNAs and its 5´-3´exonuclease cleaves TaqMan probes to generate fluorescent signals.
For the reverse transcription step, this kit uses a super-efficient Thermophilic Reverse Transcriptase (US patent pending), a thermophilic type A polymerase, which works best at 60-62°C, overcoming difficulties due to high GC content and abundant secondary structure. The RTase efficiently synthesizes a complementary DNA strands on RNA templates with 1-2 unit per 20μL of reaction. The RTase reversely-transcribes single digit copies of target RNA molecules consistently.
The kit also contains qPCR polymerase, which is specially engineered for TaqMan probes and its increased 5’-3’ exonuclease activity generating S-shaped curves.
