LEADGENE^®5X One-Step Probe RT-qPCR Master Mix (Glycerol-Free)

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Datasheet

Description
Manual

Leadgene^® 5X One-Step Probe RT-qPCR Master Mix (Glycerol-Free) 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. This product is a glycerol-free formulation.

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
5X One-Step Probe RT-qPCR Master Mix (Glycerol-Free)	4 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	95°C 5 – 15 sec
Annealing/Extension	40 – 45	55 – 65 °C	30 – 60 sec