A 2X qPCR master mix looks like a commodity until it does not match your instrument or chemistry. This guide covers dye vs probe, ROX levels, hot-start, setup volumes and how to validate efficiency.
Range: qPCR and RT-PCR reagents4 minute readUpdated
A 2X qPCR master mix, or premix, contains everything except primers, template and water, so setup is a two- or three-pipette job and reactions are consistent across a plate:
| Dye-based (SYBR Green type) | Probe-based (hydrolysis probe) | |
|---|---|---|
| Detects | Any double-stranded DNA | One sequence the probe binds |
| Specificity | Set by primers; confirm with a melt curve | Primers plus probe |
| Multiplexing | No | Yes, with different dyes |
| Cost per reaction | Lower | Higher (probe synthesis) |
| Best for | Gene expression screens, many targets | Diagnostics-style assays, SNPs, multiplex |
ROX is a passive reference dye that does not take part in the reaction. Some instruments divide each well’s signal by its ROX signal to correct for differences in volume and optics. Using the wrong level adds noise and can trigger instrument warnings.
| ROX level | Examples |
|---|---|
| High ROX | Applied Biosystems StepOne, StepOnePlus, 7000, 7300, 7900HT |
| Low ROX | Applied Biosystems 7500, ViiA 7, QuantStudio series |
| Not needed | Bio-Rad CFX, Roche LightCycler, Qiagen Rotor-Gene |
At room temperature, primers can bind loosely to each other and to off-target sequences, and an active polymerase extends them, making primer-dimers and non-specific products that compete with your target and, in dye-based qPCR, add signal. Hot-start polymerases are blocked by an antibody, a chemical modification or an aptamer until the first denaturation releases them.
Antibody-based hot-start activates in a minute or two; chemically modified enzymes need longer, often 10 minutes or more. Fast-cycling mixes shorten each cycle so a 40-cycle run takes well under an hour on a compatible instrument.
| Component | Volume | Final |
|---|---|---|
| 2X master mix | 10 µL | 1X |
| Forward primer, 10 µM | 0.4–0.8 µL | 200–400 nM |
| Reverse primer, 10 µM | 0.4–0.8 µL | 200–400 nM |
| Template | 1–2 µL | |
| Nuclease-free water | To 20 µL |
Make a master mix of everything except template for all wells plus about 10%, spin the plate briefly before loading, and run three technical replicates per sample.
Run a five-point, ten-fold dilution series of template and plot Cq against log input. Efficiency is calculated from the slope:
Efficiency = 10(−1/slope) − 1. A slope of −3.32 means 100%, a perfect doubling each cycle. Accept 90–110% with R² of 0.98 or more.
For dye-based assays, a single sharp peak in the melt curve shows one product. A second, lower-temperature peak usually means primer-dimers, which the no-template control will confirm.
For how qPCR fits with reverse transcription and end-point PCR, see the PCR vs qPCR vs RT-qPCR guide. The qPCR and RT-PCR range lists the premixes, Taq and reverse transcription kits.
No. The dye binds all double-stranded DNA and would swamp the probe signal. Use a mix made for probes, which has no intercalating dye.
Usually the run still works, but normalisation is off, which adds noise between wells and can trigger warnings in the instrument software. Use the level your instrument expects.
Between 90% and 110%, from a standard curve with a slope between about -3.6 and -3.1 and R² of at least 0.98. Efficiencies outside that range point to poor primers, inhibitors or pipetting error.
In dye-based qPCR, a late peak at a lower melt temperature is usually primer-dimer. A peak matching your product means contamination: replace reagents, clean the setup area and keep amplified products away from setup.
For research use only. Protocols here are general outlines: follow your kit manual and your institution’s safety rules. See our editorial policy.
Products for this workflow
Taq DNA polymerase, PCR and qPCR premixes, and reverse transcription kits for cDNA synthesis.
qPCR / RT-PCR
End-point PCR tells you whether a sequence is there, qPCR tells you how much, and RT-qPCR does the same for RNA. Here is what changes between them and the reagent list for each.
4 minute read
Molecular biology
Cloning is a chain of small steps, and most failures trace back to one of them. Here is the whole chain, the reagent each step needs, and how to check you have the right clone before you build on it.
4 minute read
qPCR / RT-PCR
Your controls usually say what went wrong. A positive control, a no-template control, a no-RT control and one dilution of the sample separate template, inhibition and primer problems.
3 minute read
Send the product names or catalogue numbers, quantities and delivery country. We'll confirm availability, lead time and the documents for qpcr and rt-pcr reagents.