DNA Amplification with ProbeMaster® Lyo ROX qPCR Master Mix

ProbeMaster® Lyo ROX is a lyophilized master mix containing all components required for quantitative PCR using intercalating dyes or hydrolysis probes. The formulation is optimized to deliver reliable results with low threshold cycle values and a high signal-to-background ratio. The ready-to-use format minimizes the risk of sample contamination.

The ProbeMaster® Lyo ROX master mix enables a wide range of applications while reducing preparation time. Due to the presence of the ROX reference dye, the mix is suitable for accurate quantification of DNA template in samples and can be used for quantitative analysis, gene expression studies, genotyping, SNP detection, and other applications.

Master Mix Composition

HS Taq DNA polymerase
Deoxynucleoside triphosphate (dNTP) mixture
PCR buffer (contains Mg²⁺)
ROX reference dye
Lyophilization stabilizers

Key features

One tube of the lyophilized mix, after reconstitution in 450 µL of water, is sufficient for 100 reactions of 25 µL each.
The mix is fully ready to use. Only the DNA sample, primers, and water need to be added to set up the reaction, which significantly reduces preparation time. The ready-to-use format also lowers the risk of contamination.
The following templates can be used: genomic DNA, viral DNA, plasmid DNA, cDNA obtained after reverse transcription, and others.
The mix contains a high-processivity Hot-Start Taq polymerase that is activated by incubation for 5 minutes at 95 °C. The HS Taq DNA polymerase is supplied as a complex of monoclonal antibodies with the enzyme. Heating during the first PCR cycle inactivates the antibodies and activates the enzyme. This Hot-Start technology prevents nonspecific amplification and primer-dimer formation.
HS Taq DNA polymerase exhibits 5′–3′ polymerase and 5′–3′ exonuclease activities. It also has terminal transferase activity, adding an extra adenine residue to the 3′ ends of double-stranded DNA, enabling the use of PCR products for TA cloning.
The mix includes the ROX reference dye for signal normalization. The ROX concentration is optimized for compatibility with most real-time PCR amplifiers.
The mix does not contain UDG or dUTP.

Possible applications

Real-time PCR
PCR with electrophoretic detection
PCR with cDNA samples after reverse transcription
Genotyping
Colony PCR

Equipment compatibility

Compatible with PCR thermocyclers of any type.

Protocol

Before starting, add 450 µL of deionized water to the lyophilized mix, wait 1 minute, vortex the tube to mix thoroughly, and briefly centrifuge to collect the contents. The reconstituted mix can be stored at 4 °C for up to 30 days or frozen at −20 °C for the product's shelf life. Up to 5 freeze–thaw cycles are allowed.

Mix the tube contents thoroughly and briefly centrifuge.
Prepare the reaction mixture according to the table below in the indicated order, calculated for (N + 0.1N) reactions, where N is the required number of reactions. Mix the prepared master mix thoroughly and briefly centrifuge.
Note: For reproducible PCR results, it is recommended to run each DNA sample in at least two replicates.

Reaction setup for one 25 µL reaction (real-time detection)

Component	Volume	Notes
PCR Master Mix, 5×	5 µL
Forward primer	0.5–1.0 µL (10 µM)	Final concentration 200–400 nM
Reverse primer	0.5–1.0 µL (10 µM)	Final concentration 200–400 nM
Probe or	0.25–0.75 µL (10 µM)	2.5–7.5 pmol per reaction (final concentration 100–300 nM)
Intercalating dye	According to manufacturer's recommendation
Deionized water	To 25 µL total volume*	Given the volume of the DNA sample to be added in step 4
DNA	2–9 µL (cDNA, 50–100 ng genomic DNA, or 1–100 pg plasmid DNA)	Add separately to each PCR tube in step 4
Total reaction volume:	25 µL*	If a different reaction volume is used, recalculate the volumes of the reaction components while maintaining the given proportions

* Reaction volume may be adjusted depending on the application, but volumes below 10 µL are not recommended.

Add the prepared master mix to PCR tubes without the DNA template volume.
Add 2–9 µL of DNA or cDNA sample to each tube using a separate pipette tip. After adding DNA, the final reaction volume should be 25 µL. Close the tube caps and briefly centrifuge.

Perform DNA amplification using the programs below (primer annealing temperature should be optimized for each primer pair).

If primer annealing temperature ≥ 60 °C

Stage	Temperature	Time	Cycles
HS Taq activation	95 °C	5 min	1
Denaturation	95 °C	10 s	40–50
Annealing combined with elongation (fluorescence detection should occur at this step**)	60–72 °C	30–60 s	40–50

If primer annealing temperature < 60 °C

Stage	Temperature	Time	Cycles
HS Taq activation	95 °C	5 min	1
Denaturation	95 °C	10 s	40–50
Annealing (fluorescence detection should occur at this step**)	55–59 °C	10–15 s
Elongation	72 °C	15–30 s

** Select the ROX channel as the passive reference.

If an intercalating dye is used, it is recommended that the amplicon be melted between 60 °C and 95 °C after amplification to ensure no nonspecific amplification.
For gel electrophoresis analysis, mix PCR products with loading buffer, load them into gel wells, and perform electrophoresis.
PCR products may be stored at −20 °C if necessary.

Storage conditions

12 months (from delivery) at temperatures up to 4 °C. Transportation: up to 21 days at temperatures up to 25 °C
After reconstitution, store at +4 °C for up to 30 days, or at −20 °C within the product shelf life. The reconstituted mixture may undergo up to five freeze-thaw cycles.
Shelf life: 12 months from the date of delivery unless otherwise specified in the product certificate.