Leadgene® 5X One-Step Probe RT-qPCR Master Mix is a one-step real-time reverse transcription-polymerase chain reaction (RT-qPCR) kit developed for cDNA synthesis and real-time PCR in the same tube. This product is suitable for probe-based detection and formulated as a 5-fold premix. Reaction can be simply set up by adding the RNA template, primers, and probes. This master mix does not contain ROX reference dye; it offers great
convenience and minimizes the risk of cross-contamination.
Stored at -20°C.
Avoid repeated freeze/thaw cycles.
Final concentrations of 400 nM (each primer) are suitable for most reactions. To obtain
optimal condition, primer concentration can be titrated between 0.2-1 μM.
A final concentration of 200 nM (probe) is suitable for most reactions. To obtain optimal
condition, probe concentration can be titrated between 0.1-0.3 μM.
To obtain optimal condition, annealing/extension temperature can be adjusted between 55°
C-65°C, annealing/extension time can be extended up to 60 sec.
Appropriate amplicon length should be arranged between 80-200 bp.
The following procedure is a general guideline for One-step RT-qPCR reaction. To
maintain an RNase-free environment, always wear disposable gloves, and use laboratory
consumables and water of nuclease-free grade during the whole experiment course.
RT-qPCR reaction set-up:
1. Place all required reagents on ice.
|5X One-Step Probe RT-qPCR
|Forward primer (10 μM)||0.8 μL||0.4 μM|
|Reverse primer (10 μM)||0.8 μL||0.4 μM|
|Probe (10 μM)||0.4 μL||0.2 μM|
|RNA template||X μL||≦ 1 μg (total RNA)|
|Nuclease-Free H2O||Y μL||-|
|Total reaction volume||20 μL||-|
* See Usage Notes for additional guidelines on primer/template preparation.
2. Gently mix the reaction thoroughly to achieve uniform distribution and briefly centrifuge.
3. Thermal cycling conditions for standard qPCR
|Reverse transcription||1||50°C||10 - 15 min|
|Enzyme activation||1||95°C||5 min|
|Denaturation||40 – 45||95°C||5 – 15 sec|
|Annealing/Extension||55 – 65 °C||30 – 60 sec|