qPCR with probes .
Our primaQUANT PROBE qPCR Master Mixes are extremely fast and deliver reproducible results even under difficult conditions.
The primaQUANT PROBE Master Mixes can be pipetted without refrigeration and are dyed blue as standard for perfect visual control during pipetting.
The primaQUANT Probe Master Mixes can be used with all common probe systems and are also ideally suited for multiplex assays.
Hard Facts .
- Excellent sensitivity < 1pg cDNA
- Ultra-Fast: qPCR in 15 minutes
- High temperature stability
- Stable at room temperature - pipetting without ice
- For better handling optionally dyed lierferable
- Suitable for multiplexing
- Made in Germany
Applications .
Our primaQUANT Probe qPCR Master Mixes are suitable for all common types of probe assays:
- Primer probes
- Hydrolysis probes (TaqMan, MGB-TaqMan, Snake Assay)
- Hybridization probes (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing);
- Nucleotide analogs (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon).
More information can be found in the following review:
Navarro, E., Serrano-Heras, G., Castaño, M. J., & Solera, J. (2015). Real-time PCR detection chemistry. Clinica Chimica Acta, 439, 231-250. https://doi.org/10.1016/j.cca.2014.10.017
The primaQUANT Probe qPCR Master Mix is perfect for genotyping using probes. Multiplexing allows several markers to be detected in parallel.
Matsuda, K. (2017). PCR-Based Detection Methods for Single-Nucleotide Polymorphism or Mutation: Real-Time PCR and Its Substantial Contribution Toward Technological Refinement. Advances in Clinical Chemistry, 80, 45-72. https://doi.org/10.1016/bs.acc.2016.11.002
To determine the expression of a gene (gene or RNA expression), the total RNA must first be transcribed into cDNA.
If you want to perform cDNA synthesis in a separate step, our primaREVERSE RT kit first choice.
Alternatively, you can also transfer the RNA directly into our primaQUANT 1STEP RT-qPCR Kit.
Multiplex qPCR assays use multiple target-specific probes in parallel, each labeled with a different fluorescent dye. In this way, the expression values of several targets or genes can be determined in parallel in a time-saving manner. This elegant method is widely used, especially in diagnostics.
Hawkins, S. F. C., & Guest, P. C. (2017). Multiplex Analyses Using Real-Time Quantitative PCR. In P. C. Guest (Ed.), Multiplex biomarker techniques: methods and applications (pp. 125-133). Springer. https://doi.org/10.1007/978-1-4939-6730-8_8
Quantitative PCR is also frequently used for the diagnosis of pathogens.
DNA pathogens (DNA viruses, bacteria, fungi) can thus be analyzed directly. For RNA viruses, e.g. SARS-COV2, a transcription of RNA into cDNA is necessary. Our primaREVERSE RT cDNA Synthesis Kit is ideally suited for this purpose. You can also use our primaQUANT 1-STEP Master Mix, which does cDNA synthesis and qPCR in one step.
Because of their high purity, our primaQUANT Master Mixes are ideal for detecting or ruling out contamination of a product with nucleic acids or nucleases.
Features .
For all qPCR cyclers .
Our qPCR 2x Master Mixes are suitable for all common qPCR cyclers and optionally available with ROX.
We have compiled a table for you showing whether your device uses ROX(6-carboxy- X-rhodamine) for normalization and which concentration, if any, is required.
qPCR in less than 20 minutes .
Extremely fast
The primaQUANT qPCR Master Mixes cope with the shortest cycle times due to their ultra fast DNA polymerase: a complete qPCR run takes less than 20 minutes under suitable conditions.
Of course, you can also use your previous qPCR protocol.
Standard protocol:
Schritt | Zeit | Temperatur | |
---|---|---|---|
Initiale Denaturierung | 1-3 Minuten | 92°C – 95°C | |
25 – 40 Zyklen | Denaturierung | 5-10 Sekunden | 92°C – 95°C |
Kombiniertes Annealing/Elongation | 10-20 Sekunden | 60°C – abhängig von den verwendeten Primern |
Fast protocol:
Schritt | Zeit | Temperatur | |
---|---|---|---|
Initiale Denaturierung | 1 Minute | 92°C – 95°C | |
25 – 40 Zyklen | Denaturierung | 5 Sekunden | 92°C – 95°C |
Kombiniertes Annealing/Elongation | 10 Sekunden | 60°C – abhängig von den verwendeten Primern |
Ultra Fast Protocol:
Highly Specific .
Our primaQUANT PROBE qPCR Master Mixes work very reliably under a wide range of assay conditions.
They are very robust with respect to primer concentration and require extremely little sample material.
Excellent performance .
Convincing values for all important performance parameters guarantee reliable and reproducible results even under difficult conditions.
- Early cq values
- Wide Dynamic Range
- Low cv values
- Low level of detection
- Detection limit < 1 pg human cDNA
Highest sensitivity of 0.1 pg cDNA input amount
No template inhibition
Wide Dynamic Range with excellent efficiency.
Always stable .
Freeze/thaw
Our PCR and qPCR reagents are not affected by multiple freezing/thawing . You can therefore freeze and thaw the mixes more than 10 times without any problems.
Refrigerator storage
The primaQUANT Master Mixes are stable for weeks at 4°C. For short-term storage, we generally recommend storage at 4°C in the refrigerator.
Stability at room temperature
The primaQUANT Master Mixes can even be stored for several days at room temperature without loosing quality. Therefore, you no longer have to pipette your qPCR laboriously on ice to avoid a loss of sensitivity!
Smart Package Size .
No more annoying aliquoting!
Our Master Mixes come in units of 1 ml as standard.
This is very convenient and practical, because one tube is enough for exactly one 96 well plate, two tubes for one 384 well plate. If you ever need less, you can simply store the mixes in the refrigerator for several weeks.
Customized fillings: As the manufacturer, we can of course supply you with fillings and mixtures according to your special requirements.
Made in Germany .
We develop and produce our enzymes and master mixes exclusively ourselves in Germany. You benefit from this in several ways:
- Very short delivery times
- Fast and competent support
- ISO certified production
- High-end quality control
- Customized fillings or product adaptations
primaQUANT qPCR Master Mixes for Probes .
Downloads .
Manufacturer | qPCR cycler | ROX necessary? |
---|---|---|
Applied Biosystems / Thermo Fischer | StepOne™ Real-Time PCR System | high ROX |
StepOnePlus™ Real-Time PCR System | high ROX | |
7500 Real-Time PCR System | low ROX | |
7500 Fast Real-Time PCR System | low ROX | |
7500 Fast Dx Real-Time PCR Instrument | low ROX | |
7500 Real-Time PCR System for Human Identification | low ROX | |
7300 Real-Time PCR System | high ROX | |
Viia™ 7 Real-Time PCR System | low ROX | |
7900HT Fast Real-Time PCR System | high ROX | |
OpenArray® Real-Time PCR Platform | high ROX | |
PRISM® 7000 Sequencing Detection System | high ROX | |
PRISM® 7700 Sequencing Detection System | high ROX | |
PRISM® 7900 Sequencing Detection System | high ROX | |
Gene Amp 5700 | high ROX | |
Quantstudio™ 3 / 3 FAST | low Rox | |
Quantstudio™ 5 / 5 FAST | low Rox | |
Quantstudio™ 6 | low Rox | |
Quantstudio™ 7 | low Rox | |
Quantstudio™ 12 | low Rox | |
QuantStudio™ 12K Flex system | low ROX | |
Bioneer | Exicycler™96 | no ROX, optional |
Exicycler™384 | no ROX, optional | |
Biorad | CFX96™ Real-Time PCR Detection System | no ROX |
CFX96 Touch™ Real-Time PCR Detection System | no ROX | |
CFX384 Touch™ Real-Time PCR Detection System | no ROX | |
Chromo4™ Four-Color Real-Time Detector | no ROX | |
CFX Connect™ Real-Time PCR Detection System | no ROX | |
iQ5 Real-Time PCR Detection System | no ROX | |
MiniOpticon™ Real-Time PCR Detection System | no ROX | |
Opticon 2 – Continuous Fluorescence Detection System | no ROX | |
Chromo4™ Four-Color Real-Time Detector | no ROX | |
Bioron | RealLine Cycler 48-4 | no ROX, optional |
RealLine Cycler 48-5 | no ROX, optional | |
RealLine Cycler 96-4 | no ROX, optional | |
RealLine Cycler 96-5 | no ROX, optional | |
DNA Technology | Dtlite | no ROX, optional |
Dtprime | no ROX, optional | |
Eppendorf | Mastercycler® ep realplex 4, Real-Time Thermal Cycler | no ROX |
Mastercycler® ep realplex 4s, Real-Time Thermal Cycler | no ROX | |
Mastercycler® ep realplex, Real-Time Thermal Cycler | no ROX | |
Mastercycler® ep realplex s, Real-Time Thermal Cycler | no ROX | |
Mastercycler Nexus | no ROX | |
Mastercycler Nexus gradient | no ROX | |
Mastercycler Pro | no ROX | |
Mastercycler Pro S | no ROX | |
Mastercycler Pro 384 | no ROX | |
Roche | LightCycler® 96 System | no ROX |
LightCycler® 480 System | no ROX | |
LightCycler® 2.0 Instrument | no ROX | |
LightCycler® 1.5 Instrument | no ROX | |
LightCycler® 1536 System | no ROX | |
LightCycler® Nano System | no ROX | |
Analytik Jena | qTower | low ROX (optional) |
qTower 2.0 | low ROX (optional) | |
qTower 2.2 | low ROX (optional) | |
qTower 3 | low ROX (optional) | |
qTower 3 84 | low ROX (optional) | |
Stratagene / Agilent | Mx3000P® qPCR System | low ROX |
Mx3005P® qPCR System | low ROX | |
Mx4000® qPCR System | low ROX | |
Qiagen | Rotor-Gene™ Q | low ROX |
Rotor-Gene™ 6000 | low ROX | |
Quantabio | Q | no ROX, optional |
Composition
Component | Stock concentration | 20 µl reaction | 10 µl reaction | Final concentration |
---|---|---|---|---|
primaQUANT Master Mix | 2x | 10 µl | 5 µl | 1 x |
Reverse primer | 4 µM | 1 µl | 0.5 µl | 200 nM (100 – 400 nM recommended) |
Forward primer | 4 µM | 1 µl | 0.5 µl | 200 nM (100 – 400 nM recommended) |
Probe | 8 µM | 1 µl | 0.5 µl | 400 nM (200 – 600 nM recommended) |
Template (DNA/cDNA) | – | variabel | variabel | 0.1 – 10 ng/reaction |
Sterile water | – | up to 20 µl | up to 10 µl | – |
Standard protocol
3-Step
Step | Time | Temperature | |
---|---|---|---|
Initial denaturation | 1-3 minutes | 92°C – 95°C | |
25 – 40 cycles | Denaturation | 5-10 seconds | 92°C – 95°C |
Annealing | 1-5 seconds | 60°C – depending on used primers | |
Extension | 10-20 seconds | 72°C |
2-Step
Step | Time | Temperature | |
---|---|---|---|
Initial denaturation | 1-3 minutes | 92°C – 95°C | |
25 – 40 Cycles | Denaturation | 5-10 seconds | 92°C – 95°C |
Combined Annealing/Elongation | 10-20 seconds | 60°C – depending on used primers |
Ultra-FAST protocol
3-Step
Step | Time | Temperature | |
---|---|---|---|
Initial denaturation | 1 minute | 92°C – 95°C | |
25 – 40 cycles | Denaturation | 1-5 seconds | 92°C – 95°C |
Annealing | 1-5 seconds | 60°C – depending on used primers | |
Extension | 1 seconds | 72°C |
2-Step