Luna® Universal qPCR Master MixProduct information
Luna Universal qPCR Master Mix
|200 rxns ( 2 X )||
Luna Universal qPCR Master Mix
|500 rxns ( 2 X )||
Luna Universal qPCR Master Mix
|1000 rxns (10x1ml) ( 2 X )||
Luna Universal qPCR Master Mix
|2500 rxns (1x25ml) ( 2 X )||
Rapid, sensitive and precise dye-based qPCR detection and quantitation of target DNA and cDNA sequences.
Make a simpler choice
- One product per application simplifies selection
- Convenient master mix formats and user-friendly protocols simplify reaction setup
- Non-interfering, visible tracking dye helps to eliminate pipetting errors
Experience best-in-class performance
- All Luna® products have undergone rigorous testing to optimize specificity, sensitivity, accuracy and reproducibility
- Products perform consistently across a wide variety of sample sources
- A comprehensive evaluation of commercially-available qPCR and RT-qPCR reagents demonstrates superior performance of Luna products
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“Dots in Boxes" Visualization of qPCR Data
Overview of qPCR
DescriptionDye-based quantitative PCR (qPCR) uses real-time fluorescence of a double-stranded DNA (dsDNA) binding dye, most commonly SYBR® Green I, to measure DNA amplification during each cycle of a PCR. At a point where the fluorescence signal is confidently detected 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 qPCR Master Mix is an optimized 2X reaction mix for real-time qPCR detection and quantitation of target DNA sequences using the SYBR®/FAM channel of most real-time qPCR instruments. 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 with fluorescent dyes 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 and DNA template. Genomic DNA or cDNA of interest can be quantitated with Luna qPCR, and existing as well as commercial qPCR assay primer sequences can be used.
- DNA Amplification, PCR & qPCR
Properties & Usage
- Primer 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.
- Primer Concentration
For most targets, a final concentration of 250 nM (each primer) will provide optimum performance. If needed, primer concentrations can be optimized between 100–500 nM.
- 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.
- 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 before addition to qPCR.
- 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 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.
- 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 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, 0.025 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.
- 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.
Protocols, Manuals & Usage
Usage & Guidelines
Tools & Resources
Faqs & Troubleshooting
- How do I use qPCR to determine the concentration of my material?
- Can I set up my Luna® qPCR at room temperature?
- What is the difference between probe- and dye-based versions of the Luna® qPCR Mixes?
- Should I use probe- or dye-based detection for my qPCR assays?
- How should I design primers for Luna® qPCR?
- How long should my amplicon be for qPCR?
- Why is the Luna® qPCR Mix blue? Will this dye interfere with detection?
- Can I run the Luna® qPCR Mix on my qPCR instrument?
- Can I use fast cycling conditions with the Luna® qPCR Mix?
- Do I need to add ROX?
- How many dilutions should I use to make a standard curve?
- Why does NEB recommend 40-45 cycles?
- Does the Luna® qPCR Mix contain dUTP? Can I use carryover contamination prevention methods?
- Why do I have multiple peaks in my melt curve?
- How can I distinguish non-template amplification (NTC) from real products?
- Why do I see amplification curves in my NTC samples?
- What samples can be used in qPCR with the Luna® Mix?
- Can I use cDNA? Does it matter how I make it?
- How much template material can I use in Luna® qPCR?
- How much primer should I use for the Luna® Universal qPCR Master Mix?
- Can I use shorter cycling times?
- What is the fluorescent, double-stranded DNA binding dye in the Luna® qPCR master mix?
Citations & Technical Literature
Quality, Safety & Legal
Quality Assurance StatementQuality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
SpecificationsThe Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
Certificate Of AnalysisThe Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety DataSheetsThe following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
Luna® Universal qPCR Master Mix
Legal and DisclaimersThis product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
LicensesNotice to Purchaser: Nucleic-acid based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. (See Patent Nos. 5,475,096; 5,670,637; 5,696,249; 5,874,557; and 5,693,502). New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the aptamer-based Luna University qPCR Master Mix for Research Purposes Only. Commercial use of the aptamer-based Luna Universal qPCR Master Mix requires a license from New England Biolabs, Inc. Please contact [email protected] for more information.
TrademarksBIO-RAD® is a registered trademark of Bio-Rad Laboratories.
FASTSTART™ is a trademark of Roche.
GOTAQ® and PROMEGA® are registered trademarks of Promega Corporation.
POWERUP™ is a trademark of Thermo Fisher Scientific.
QIAGEN® and QUANTITECT® are registered trademarks of Qiagen, Inc.
QUANTSTUDIO® is a registered trademark of Life Technologies Corporation.
SOMAMER® is a registered trademark of Somalogic, Inc.
SSOADVANCED™ is a trademark of Bio-Rad Laboratories.
STEPONEPLUS® is a registered trademark of Applied Biosystems, LLC.
SYBR® is a registered trademark of Molecular Probes, Inc.
The supporting documents available for this product can be downloaded below.