LEADGENE^® 2X One-Step Probe RT-qPCR Master Mix

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Datasheet

Description
Manual

Leadgene® 2X 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 2-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.

Storage

Stored at -20°C.
Avoid repeated freeze/thaw cycles.

Note

Primer/Probe concentration
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.
Annealing/Extension optimization
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.
Target length
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.

Component	Amount	Final concentration
2X One-Step Probe RT-qPCR Master Mix	10 μL	1X
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

Step	Cycles	Temperature	Time
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	40-45	55 – 65 °C	30 – 60 sec