The One-Step RT-PCR Kit provides a convenient, sensitive, and reproducible method to detect and analyze the RNA molecules by reverse transcription polymerase chain reaction (RT-PCR). The components for cDNA synthesis and PCR amplification are combined in a kit, using gene-specific primers and target RNAs from either total RNA or mRNA. Reverse transcription automatically follows PCR cycling without additional steps. The kit consists of three major components: RT/ HotStar Taq Mix, 2X Reaction Mix, and 50 mM Magnesium Sulfate (MgSO4). The RT/ HotStar Taq Mix contain a mixture of Reverse
Transcriptase (RTase) and HotStar Taq DNA polymerase for optimal cDNA synthesis and PCR amplification. The RTase is modified from the Moloney Murine Leukemia Virus (M-MLV) RTase, engineered to reduce RNase H activity and increase thermal stability. The HotStar Taq DNA polymerase is Taq DNA polymerase complexes with a proprietary antibody that blocks activity at ambient temperatures. Activity is restored after the enzyme activation step at 95°C, thereby providing an automatic “hot-start” for Taq DNA polymerase in PCR, increasing the sensitivity and specificity of PCR reaction. The 2X Reaction Mix
consists of 6 mM MgSO4, deoxyribonucleotide triphosphates (dNTPs), and stabilizers provide a proprietary buffer system optimized for reverse transcription and PCR amplification. One addition tube of 50 mM MgSO4 is included in the kit. The 2X Reaction Mix is well for most targets; however, the optimal concentration may range from 3 to 6 mM. If necessary, use the separate 50 mM MgSO4 buffer to increase the magnesium concentration.
|2X Reaction Mix|
|50 mM Magnesium Sulfate|