Luna® Universal Probe qPCR Master Mix



Rapid, sensitive and precise probe-based qPCR detection and quantitation of target DNA and cDNA sequences.

Probe-based quantitative PCR (qPCR) uses real-time fluorescence released upon 5´→3´ exonuclease cleavage of a quenched, target-specific probe to measure DNA amplification at each cycle of a PCR. At a point where the fluorescence signal is significantly detectable over the background fluorescence, a quantification cycle or Cq value can be determined. Cq values can be used to evaluate relative target abundance between two or more samples or to calculate absolute target quantities in reference to an appropriate standard curve, derived from a series of known dilutions.

The NEB Luna Universal Probe qPCR Master Mix is a 2X reaction mix optimized for real-time qPCR detection and quantitation of target DNA sequences using hydrolysis probes. It contains Hot Start Taq DNA Polymerase and has been formulated with a unique passive reference dye that is compatible across a variety of instrument platforms (including those that require a high or low ROX reference signal). It also features dUTP for carryover prevention and a non-fluorescent, visible dye to monitor reaction setup. This dye does not spectrally overlap fluorophores commonly used for qPCR and will not interfere with real-time detection.

The master mix formulation is supplied at 2X concentration and contains all PCR components required for amplification and quantitation of DNA except primers/probes and DNA template. Genomic DNA or cDNA of interest can be quantitated with Luna qPCR and existing as well as commercial qPCR assay primer/probe sequences can be used.

Figure 1: NEB’s Luna Universal Probe qPCR Master Mix offers exceptional sensitivity, reproducibility and qPCR performance
qPCR targeting human GAPDH was performed using the Luna Universal Probe qPCR Master Mix over a 6-log range of input template concentrations (20 ng – 0.2 pg Jurkat-derived cDNA) with 8 replicates at each concentration. cDNA was generated from Jurkat total RNA using the NEB Protoscript® II First Strand cDNA Synthesis Kit (NEB #E6560).

Figure 2: NEB’s Luna Universal Probe qPCR Master Mix offers robust performance in multiplex applications
Singleplex (left) and multiplex (right) qPCRs targeting human GAPDH, ribosomal protein L32g and PI-3-Kinase-Related Kinase SMG1 were performed using the Luna Universal Probe qPCR Master Mix over a 5-log range of input template concentrations (20 ng – 2 pg Jurkat-derived cDNA) with 4 replicates at each concentration. 0.4 µM primer was used for the lower-copy target (SMG1) and 0.2 µM primer for each higher-copy target (L32g and GAPDH), in both multiplex qPCR (to account for copy number differences) and singleplex qPCR (to allow direct comparison).

Figure 3: Extensive performance evaluation of commercially available probe-based qPCR reagents demonstrates the robustness and specificity of Luna
Luna DNA probe
qPCR reagents from NEB and other manufacturers were tested on 10 qPCR targets, varying in abundance, length and % GC, using either Jurkat genomic DNA or Jurkat-derived cDNA as input (5 targets each). For each testing condition, data was collected by 2 users and according to manufacturer specifications. Results were evaluated for efficiency, low input detection and lack of non-template amplification (where ΔCq = average Cq of non-template control – average Cq of lowest input). In addition, consistency, reproducibility and overall curve quality were assessed (Quality Score). Bar graph indicates % of targets that met acceptable performance criteria (indicated by green box on dot plot and Quality Score > 3). Results for NEB and other major manufacturers are shown: QIAGEN, QuantiTect® Probe PCR Kit; Bio-Rad, SsoAdvanced Universal Probes Supermix; Roche, FastStart® TaqMan® Probe Master; ABI, TaqMan Fast Advanced Master Mix; Promega®, GoTaq® Probe qPCR Master Mix. NEB’s Luna Universal Probe qPCR Master Mix outperformed all other reagents tested.

Learn more about our comprehensive qPCR/RT-qPCR testing and “dots in boxes” data visualization

Properties and Usage

Storage Temperature



  1. Assay Design
    The use of qPCR primer design software (e.g., Primer3) maximizes the likelihood of amplification success while minimizing nonspecific amplification and primer dimers. Targets with balanced GC/AT content (40–60%) tend to amplify efficiently. Where possible, enter sufficient sequence around the area of interest to permit robust primer design and use search criteria that permit cross-reference against relevant sequence databases (to avoid potential off-target amplification). For cDNA targets, it is advisable to design primers across known splicing sites in order to prevent amplification from genomic DNA. Conversely, primers designed to target intronic regions can ensure amplification exclusively from genomic DNA.
  2. Primer and Probe Concentration
    For most targets, a final concentration of 400 nM for each primer will provide optimum performance. If needed, primer concentrations can be optimized between 200–900 nM. Probe should be included at 200 nM for best results. Probe concentration can be optimized in the range of 100–500 nM if optimization of performance or target fluorescence level is desired.
  3. Multiplexing
    To detect or quantitate multiple targets in the same Luna reaction, select different fluorophores corresponding to separate detection channels of the real-time instrument. Include 400 nM of forward and reverse primer and 200 nM probe for each target to be detected in the reaction, and adjust concentrations if necessary based on performance (primer 200–900 nM, probe 100–500 nM). When loading qPCR protocol onto the real-time instrument, be sure to select the appropriate optical channels, as some instruments have a single channel recording mode that would prevent multiplex data collection and analysis. For ROX-dependent instruments, avoid ROX-labeled probes. The functionality of the primer and probe sets should be tested individually before attempting a multiplex reaction. When determining which fluorophores to include in a multiplex reaction, be sure to choose compatible reporter dyes and quenchers that have well separated fluorescence spectra or exhibit minimal overlap.
  4. Amplicon Length
    To ensure successful and consistent qPCR results, it is important to maximize PCR efficiency. An important aspect of this is the design of short PCR amplicons (typically 70–200 bp). Some optimization may be required (including the use of longer extension times), for targets that exceed that range.
  5. Template Preparation and Concentration
    Luna qPCR is compatible with DNA samples prepared through typical nucleic acid purification methods. Prepared DNA should be stored in an EDTA-containing buffer (e.g., 1X TE) for long-term stability, and dilutions should be freshly prepared for a qPCR experiment by dilution into either TE or water.
    Generally, a useful concentration of standard and unknown material will be in the range of 106 copies to 1 copy. For gDNA samples from large genomes (e.g., human, mouse) a range of 50 ng–1 pg of gDNA is typical. For small genomes, adjust as necessary using 106 –1 copy input as an approximate range. Note that for single copy dilutions, some samples will contain multiple copies and some will have none, as defined by the Poisson distribution.
    For cDNA, use the product of a reaction containing 1 μg–0.1 pg starting RNA. cDNA does not need to be purified before addition to the Luna reaction but should be diluted at least 1:10 into the qPCR.
  6. ROX Reference Dye
    Some real-time instruments recommend the use of a passive reference dye (typically ROX) to overcome well-to-well variations that could be caused by bubbles, small differences in volume, and autofluorescence from dust or particulates in the reaction. The Luna Universal Probe qPCR Master Mix is formulated with a universal reference dye that is compatible with a variety of qPCR instrument types, including those that use no passive reference normalization and those that use a low or high concentration of passive reference dye (ROX). Therefore, no additional components are required to ensure compatibility with these instruments.
  7. Carryover Contamination Prevention
    qPCR is an extremely sensitive method, and contamination in new qPCR assays with products from previous amplification reactions can cause a variety of issues such as false positive results and a decrease in sensitivity. The best way to prevent this “carryover” contamination is to practice good laboratory procedures and avoid opening the reaction vessel post amplification. However, to accommodate situations where additional anti-contamination measures are desired, the Luna Universal Probe qPCR Master Mix contains a mixture of dUTP/dTTP that results in the incorporation of dU into the DNA product during amplification. Pretreatment of qPCR experiments with uracil DNA glycosylase (UDG) will eliminate previously-amplified uracil-containing products by excising the uracil base to produce a non-amplifiable DNA product. The use of a thermolabile UDG is important, as complete inactivation of the UDG is required to prevent destruction of newly synthesized qPCR products.

    To enable carryover prevention, 0.2 units/μl Antarctic Thermolabile UDG (NEB #M0372) should be added to the reaction mix. To maximize elimination of contaminating products, set up the qPCR experiments at room temperature or include a 10 minute incubation step at 25°C before the initial denaturation step.
  8. Reaction Setup and Cycling Conditions
    Due to the hot start nature of the polymerase, it is not necessary to preheat the thermocycler prior to use or set up reactions on ice.
    For 96-well plates, we recommend a final reaction volume of 20 μl.
    For 384-well plates, a final reaction volume of 10 μl is recommended.
    When programming instrument cycling conditions, ensure a plate read is included at the end of the extension step, and a denaturation (melt) curve after cycling is complete to analyze product specificity.
    Amplification for 40 cycles is sufficient for most applications, but for very low input samples 45 cycles may be used.


  1. How do I use qPCR to determine the concentration of my material?
  2. Can I set up my Luna® qPCR at room temperature?
  3. What is the difference between probe- and dye-based versions of the Luna® qPCR Mixes?
  4. Should I use probe- or dye-based detection for my qPCR assays?
  5. How should I design primers for Luna® qPCR?
  6. How long should my amplicon be for qPCR?
  7. Why is the Luna® qPCR Mix blue? Will this dye interfere with detection?
  8. Can I run the Luna® qPCR Mix on my qPCR instrument?
  9. Can I use fast cycling conditions with the Luna® qPCR Mix?
  10. Do I need to add ROX?
  11. How many dilutions should I use to make a standard curve?
  12. Why does NEB recommend 40-45 cycles?
  13. Does the Luna® qPCR Mix contain dUTP? Can I use carryover contamination prevention methods?
  14. Can I run multiplex reactions with the Luna® Universal Probe Master Mix? Do I need to change my reaction conditions?
  15. Can I use a ROX-labeled probe with the Luna® Probe Mix?
  16. Can alternative probe based detection strategies be used with the Luna® Probe Mix?
  17. What samples can be used in qPCR with the Luna® Mix?
  18. Can I use cDNA? Does it matter how I make it?
  19. How much template material can I use in Luna® qPCR?
  20. How much primer and probe should I use with the Luna® Universal Probe qPCR Master Mix?
  21. Can I use shorter cycling times?


  1. Luna® Universal Probe qPCR Master Mix Protocol (M3004)


The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

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