KiCqStart® SYBR® Green qPCR ReadyMix™ is a 2X concentrated, ready-to-use reaction cocktail that contains all components, except primers and template, for real-time quantitative PCR (qPCR). This unique combination of proprietary buffer, stabilizers, and KiCqStart® Taq DNA polymerase delivers maximum PCR efficiency, sensitivity, specificity and robust fluorescent signal using fast, or conventional cycling protocols with SYBR® Green qPCR.
Highly specific amplification is crucial to successful qPCR with SYBR® Green I dye technology because this dye binds to and detects any dsDNA generated during amplification. KiCqStart® Taq DNA polymerase contains a proprietary mixture of monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step (> 48 hours at room temperature). Activation of the enzyme is instantaneous at 95 °C. Replication of fragments up to 200 bp is complete in less than 20s at 60 °C.
For additional information, please see the qPCR Technical Guide or SYBR® Green I-based qPCR technical animation.
Different real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is critical to match the appropriate qPCR reagent to your specific instrument. KiCqStart® SYBR® Green qPCR ReadyMix™ does not contain an internal reference dye. Please consult the following table to find the optimal kit for your instrument platform.
* Full activation of KiCqStart Taq DNA polymerase occurs within 1 second at 95 ºC; however, optimal initial denaturation time is template dependent and will affect qPCR efficiency and sensitivity. Amplification of genomic DNA or supercoiled plasmid DNA targets may require 5 to 10 min at 95 ºC to fully denature and fragment the template. Short double-stranded DNA template (PCR product) or single-stranded DNA template, may require as little as 1sec at 95 ºC. Use 30 sec at 95 ºC as a general starting point.
† Extension time is dependent upon amplicon length and minimal data collection time requirement for your qPCR instrument. Some primer sets may require a 3-step cycling protocol for optimal performance. Optimal annealing temperature and time or primer concentration may need to be empirically determined for any given primer set and real-time instrument.