2x SYBR® Green I Hot-Start real-time PCR-Mix is a 2x-concentrated, ready-to-use reaction cocktail containing all components, except primers, for the amplification and detection of DNA in real-time quantitative PCR with intercalating dye SYBR® Green I.
Recombinant Q-Therm™ Taq DNA polymerase is a variant of Taq DNA polymerase with a genetically modified pol gene providing a Hot-Start for PCR. The modification renders the enzyme inactive at 37°C. At temperatures over 68°C the enzyme restores its activity. Pre-heating at 95°C is not needed to activate this enzyme.
Store 2x SYBR® Green I Hot-Start real-time PCR-Mix at -15 to -25°C. For short-term storage the master mix may be stored at +2 to +8°C. Keep the master mix away from light. Avoid repeated freezing and thawing.
2X PCR MIX COMPOSITION
Q-Therm™ Taq DNA polymerase 0.05 units/µl
20 mM Tris-HCl pH 8.8 (at 25ºC)
110 mM KCl
5 mM MgCl2
dNTPs 0.4 mM of each
SYBR® Green I
2x 1.25 ml (for 100 reactions in 50 µl assay)
For 25 µl real-time PCR reaction add the following components:
Amplification protocol (for Bio-Rad iCycler):
An example of the quantitative PCR with human PII gene primers and the standard curve construction:
Amplification protocol (Bio-Rad iCycler):
PCR amplification curve: