Applied Biosystems™ TaqMan™ Fast Advanced Master Mix for qPCR

Catalog No.	Quantity
44-445-56	1 x 1 mL
44-445-57	1 x 5 mL
44-449-63	2 x 5 mL
44-449-64	5 x 5 mL
44-449-65	10 x 5 mL
44-445-58	1 x 50 mL

Catalog No. 44-445-56 Supplier Applied Biosystems™ Supplier No. 4444556

Description

TaqMan Fast Advanced Master Mix delivers accurate quantification and dependability in less time on an array of qPCR instrument platforms.

TaqMan Fast Advanced Master Mix delivers accurate quantification and dependability in less time on an array of qPCR instrument platforms. It provides best in class performance, including the widest quantifiable range, in both single and duplex reactions, even with challenging targets. TaqMan Fast Advanced Master Mix contains AmpliTaq Fast DNA Polymerase, uracil-N-glycosylase (UNG), dNTPs with dUTP, ROX dye (passive reference), and optimized buffer components. It is supplied at a 2X concentration.

Features of the TaqMan Fast Advanced Master Mix include:

• Best-in-class performance—superior sensitivity, accuracy, dynamic range, and specificity compared to standard mixes in standard mode
• Engineered for enhanced benchtop stability—stable at room temperature for up to 72 hours in preassembled reactions
• Optimized for multiplexing—validated for duplexing with exogenous and endogenous internal positive control assays
• Reduced run times (<40 minutes) on fast and standard instrumentation—optimized for fast mode on fast instruments and fast cycling conditions on standard instruments
• Seamlessly transitions into your experiments—validated with TaqMan assays for gene expression and microRNAs, and TaqMan array micro-fluidic cards

Best-in-class performance

TaqMan Fast Advanced Master Mix has been designed to provide performance equal to or better than standard master mixes. It has been benchmarked against the leading suppliers' standard and fast master mixes to help ensure that it succeeds in providing best-in-class sensitivity, accuracy, dynamic range, and specificity. TaqMan Fast Advanced Master Mix's dynamic range (up to 7 logs) is the widest in the industry. The impressive sensitivity of the mix is showcased in the figure below which compares the Ct values of TaqMan Fast Advanced Master Mix and TaqMan Universal PCR Master Mix across a panel of gene expression assays.

Benchtop stability for high-throughput handling and convenience

TaqMan Fast Advanced Master Mix has been engineered to retain a high level of performance in preassembled reactions for up to 72 hours. The stability of this mix provides users of high-throughput liquid handling systems the assurance that the results on the first plate will mimic those of the last plate. The figure below shows an assay that was run upon assembly (time 0) and after 72 hours of incubation at 30°C, simulating the most extreme room temperature scenario.

For those with less extreme throughput needs, the enhanced stability of this master mix provides an overall added convenience to your workflow, as you are no longer constrained to immediately running plates upon assembly.

Optimized for multiplexing

We realize that confidence is paramount when it comes to your results. For added confidence in the results in every well, TaqMan Fast Advanced Master Mix has been designed to help deliver accurate results for duplex reactions using an internal positive control (IPC). The figure below shows results for the experimental target β-actin (gene name: ACTB), which was serially diluted and amplified in single-target reactions and duplex reactions.

Validated for microRNA assays

TaqMan Fast Advanced Master Mix has been validated for use with multiple real-time PCR applications, including microRNA assays. The formulation provides high specificity and dynamic range, the two most critical performance attributes that define successful results when working with microRNAs. The data in the figure below demonstrate excellent PCR linearity over a 6-log range of input template.

Reduced run times on standard instrumentation

TaqMan Fast Advanced Master Mix has been optimized for use with both fast and standard instrumentation, enabling researchers who currently own standard instruments to also reap the performance benefits and time savings this mix provides. The figure below showcases the impressive results achieved when using TaqMan Fast Advanced Master Mix under fast thermal cycling conditions on the Applied Biosystems 7300 Real-Time PCR System. The mix has been tested with all Applied Biosystems standard real-time PCR instrumentation (7900HT, 7500, and 7300 systems) to enable success independent of whether or not you own a fast-enabled instrument.

Order Info

Shipping Conditions: Dry Ice

Specifications

• Concentration: 2X
• Content And Storage: Contains 1 tube (1 mL) 2X TaqMan Fast Advanced Master Mix sufficient for 100 reactions
  
  TaqMan Fast Advanced Master Mix contains AmpliTaq Fast DNA Polymerase, Uracil-N glycosylase (UNG), dNTPs (with dUTP), ROX dye (passive reference), and optimized buffer.
  
  Store at 2°C to 8°C in the dark.
• Detection Method: Primer-probe
• Form: Liquid
• GC-Rich PCR Performance: High
• PCR Method: qPCR
• Polymerase: AmpliTaq Fast DNA Polymerase
• Reaction Speed: Fast
• For Use With (Equipment): 7500 Fast System, 7500 System, 7900HT System, QuantStudio 12k Flex, QuantStudio 3, QuantStudio 5, QuantStudio 6 Flex, QuantStudio 6 Pro, QuantStudio 7 Flex, QuantStudio 7 Pro, StepOne, StepOnePlus, ViiA 7 System
• Multiplex Capability: Duplex
• Passive Reference Dye: ROX (Pre-mixed)
• Product Line: TaqMan
• Product Type: Fast Advanced Master Mix
• Quantity: 1 x 1 mL
• Shipping Condition: Wet Ice
• Target Specificity: cDNA or gDNA only
• For Use With (Application): Gene Expression, DNA Quantification, Presence/Absence, miRNA analysis
• Thermostability: 72 hr., Assembled Reaction
• Fidelity (vs. Taq): 2 X
• No. of Reactions: 100 Reactions
• Sample Type: DNA (Genomic), cDNA
• Volume: 1 mL
• Includes: dUTP, UNG/UDG
• Inhibitor Tolerance (Simple): High

Frequently Asked Questions (FAQs)

What can I do to improve the sensitivity of my qPCR assay?

If you are targeting a low-abundance gene, you may have trouble getting Ct values in a good, reliable range (Ct > 32). To increase the sensitivity of the assay, you may want to consider the following:

- Increase the amount of RNA input into your reverse transcription reaction, if possible
- Increase the amount of cDNA in your qPCR reaction (20% by volume max)
- Try a different reverse transcription kit, such as our SuperScript VILO Master Mix, for the highest cDNA yield possible
- Consider trying a one-step or Cells-to-CT type workflow (depending on your sample type)

How do I set the baseline for my qPCR experiment?

Most times your instrument software can automatically set a proper baseline for your data. Check out our short video, Understanding Baselines, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=5BjFAJHW-bE).

How do I set the threshold for my qPCR experiment?

In most cases your instrument software can automatically set a proper threshold for your data. Check out our short video, Understanding Thresholds, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=H_xsuRQIM9M).

I am not getting any amplification with my TaqMan Assay or SYBR Green primer set. What is causing this?

There could be several reasons for no amplification from an assay or primer set. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/no-amplification.html) for more details.

I am getting amplification in my no-template control (NTC) wells in my qPCR experiment. What is causing this?

There could be several reasons for amplification in a NTC well. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/amplification-no-template-control.html) for more details.

My amplification curves have a funny shape in my qPCR experiment. What is causing this?

There are several reasons that amplification could be delayed. Please see the information in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/abnormal-amplification-curves/amplification-occurs-later.html) for more details.

What can I do if the amplification of my target gene is later than expected for my qPCR experiment?

There are several reasons that amplification could be delayed. Please see the information in our Real-Time Troubleshooting Tool for more details (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/abnormal-amplification-curves/amplification-occurs-later.html).

Can I use my SYBR Green primers for a TaqMan assay?

It may be possible to use your SYBR Green primers for a TaqMan assay, depending on how they were designed. You would have to design a separate probe to use with your existing primers. Please refer to the guidelines in this manual (https://tools.thermofisher.com/content/sfs/manuals/cms_041902.pdf) on “Manually Designing Primers and Probes” for the next steps. If you have Primer Express Software, you can use that software to design a probe. Please note that restricting the design using the predesigned SYBR primers may not allow for a successful probe design.

Do I have to normalize my samples for comparative Ct experiments?

Comparative Ct experiments use an endogenous control gene to normalize the cDNA input. Please watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=jst-3hD_xFQ) for more details on how this works. For a protocol workflow, please refer to our Guide to Performing Relative Quantitation of Gene Expression (https://tools.thermofisher.com/content/sfs/manuals/cms_042380.pdf).

What are the requirements for a relative quantification qPCR experiment?

In a relative quantification experiment, you will need to identify an endogenous control and a reference (or calibrator) sample. An endogenous control is a gene that does not change in expression across all the samples in your study. A reference sample is the sample that you are comparing all others to. This is often the untreated, or control, sample. Please see our Relative Gene Expression Workflow bulletin (https://tools.thermofisher.com/content/sfs/brochures/cms_075428.pdf) for more step-by-step guidelines on how to design your experiment.

What are the requirements for a standard curve qPCR experiment?

In a standard curve experiment, you must generate a standard curve for each target gene. The standards should closely represent the sample (i.e., RNA for RNA input, plasmid or gDNA for DNA input). This reference (http://www.ncbi.nlm.nih.gov/pubmed/11013345) is a good review of standard curves and the experimental setup. You can also review this short video (https://www.youtube.com/watch?v=mE5ieko9_RQ) on standard curve experiments.

What is the difference between absolute quantification (AQ) and relative quantification (RQ)? How do I choose which method to use?

Absolute quantification will quantitate unknowns based on a known quantity. It involves the creation of a standard curve from a target of known quantity (i.e., copy number). Unknowns can then be compared to the standard curve and a value can be extrapolated. Absolute quantification is useful for quantitating copy number of a certain target in DNA or RNA samples. The result usually is a number followed by a unit, such as copy number and ng, etc.

Relative quantification can quantitate a fold difference between samples. It involves the comparison of one sample to another sample (calibrator) of significance. For example, in a drug treatment study you could compare a treated to an untreated sample. The quantity of the calibrator is not known and cannot be measured absolutely. Therefore the calibrator (untreated sample) and samples (treated samples) are normalized to an endogenous control (a gene that is consistently expressed among the samples) and then compared to each other to get a fold difference. Relative quantification is useful for quantitating messenger RNA levels. Since the result is a fold change or ratio, it is not followed by a unit.

The method that you choose will depend on the type of data you need from your experiment. You can find more information here (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html) as well.

Can I do a melt curve with a TaqMan assay?

No. A TaqMan probe, once cleaved, cannot be re-quenched. Therefore a melt curve does not apply when using a TaqMan assay.

What is the difference between TaqMan and SYBR Green methods of detection?

TaqMan and SYBR Green chemistries are two different methods of detection for qPCR. Please see this detailed comparison of these two approaches (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/taqman-assays-vs-sybr-green-dye-for-qpcr.html). You can also watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=fkUDu042xic) on how TaqMan assays work.

How many replicates do I need to run for my qPCR experiment? What recommendations do you have for plate setup?

Please view this short video (https://www.youtube.com/watch?v=eIaPGhOjBQo), which explains some best practices for replicates and plate setup.

What are the different phases of a qPCR reaction?

Check out this short video (https://www.youtube.com/watch?feature=player_embedded&v=4sXPUbIrh3A) to understand the different phases of the PCR reaction and why they are important.

I'm trying to decide between purchasing a one-step or two-step RT-PCR kit. Can you review the advantages and disadvantages of each?

One-step RT-PCR is convenient and less prone to contamination, as there is less opportunity for pipetting error. This method is also faster than the two-step process. However, the cDNA cannot be archived, and fewer genes can be analyzed. Two-step RT-PCR gives you the ability to archive cDNA, analyze multiple genes, and offers greater flexibility. Learn more about the difference between one-step and two-step RT-PCR on this page Onestep vs Twostep RT-PCR.

Can Ct's greater than a cut-off be considered valid results?

Yes they can. However, it is important to recognize the true linearity and detection limits of your assay: Ct values above the cut-off can indicate non-specific amplification, unless your NTC is a true- no-target control, and you have run a statistically significant number of replicates. Any results with Ct above the recommended cut-off need to be validated with individual assays on plates.

What is a good Ct cut-off for the TaqMan MicroRNA Array Cards and TaqMan Advanced miRNA Array Cards? In other words, beyond what Ct should I not trust the data?

The typical Ct cut-off on TaqMan Array Cards is 32, which is equivalent to Ct 35 on a plate (10 µl reaction). Previous studies show that if you use pre-amplification, a Ct cut-off of 29 or 30 can be used to reduce numbers of false positives (see Technical Note Optimized protocols for human or rodent microRNA profiling with precious samples). To ensure that you have selected a correct cut-off, you should run replicates of the same sample and use Ct cut-off before you see an increase in the Standard Deviation.

Do I have to use the Universal Master Mix with my TaqMan MicroRNA Assays? If so, which one?

Yes. This is the recommended master mix for TaqMan MicroRNA Assays. You can use Universal Master Mix or Universal Master Mix II (with or without UNG). You can also use the Fast Advanced Master Mix.

What the thermal profile should I use for the Fast Advanced Master Mix on a TaqMan Gene Expression Array card on the ViiA 7 or QuantStudio systems?

We recommend that you use the following thermal profile for array cards: 2 min at 50 degrees C, 10 min at 92 degrees C, followed by 40 cycles of 1 sec at 97 degrees C and 20 sec at 62 degrees C.

What is the advantage of using TaqMan Fast Advanced Master Mix on the 7900HT Fast System, 7500 Fast System, ViiA 7, StepOne, StepOnePlus, or QuantStudio System (Fast Mode)?

TaqMan Fast Advanced Master Mix will have overall real time PCR runs lasting about 37 to 40 minutes, which is at least 3X faster than standard runs with the standard TaqMan Universal PCR Master Mix. It has comparable results and also provides further multiplex capability.

Are there any recommendations for running primer-limited VIC dye-labeled  TaqMan Endogenous Controls using the Fast Universal PCR Master Mix such as Cat. No. 4352042?

For using primer-limited VIC dye-labeled TaqMan Endogenous Controls in a single-plex reaction, we recommend starting with an annealing/extension time of 30 sec and extension temperature of 62 ºC. For optimal performance, the recommendation is to use the TaqMan Fast Advanced Master Mix for such applications.

Are there any recommendations for running multiplex assays using the Fast Universal PCR Master Mix such as Cat. No. 4352042?

Multiplex assays applications are complex with variable performance results across different assays. Our recommendation is to use the TaqMan Fast Advanced Master Mix for such applications.

I want to purchase TaqMan Array Human MicroRNA A Cards v2.0 (Cat. No. 4398965). What other reagents do I need to purchase in addition to these cards?

In order to perform reverse transcription on these cards, you will need the following:

• TaqMan MicroRNA Reverse Transcription Kit (Cat. No. 4366596)
• Megaplex RT Primers pools (for human Type A cards Cat. No. 4399966, for Type B cards Cat. No. 4444281)

You can proceed with or without a preamplification step which depends on the total RNA amount. Total RNA amount of 1‐1000 ng supports reverse transcription reaction with preamplification, whereas 350‐1000 ng of total RNA supports a reaction without preamplification. In case you are performing preamplification you would also need the TaqMan PreAmp Master Mix (Cat. No. 4391128) and the Megaplex PreAmp Primers, Human Pool A v2.1 (Cat. No. 4399233). You will also need the Master Mix for the qPCR reaction. It is not included in the card kit. The recommended Master Mix is: TaqMan Fast Advanced Master Mix – run mode Fast (Cat. No. 4444556) For further information, please see the following link.

What products do you recommend for downstream analysis by qPCR when using the SuperScript IV Single Cell/Low Input cDNA PreAmp Kit?

We recommend using TaqMan Fast Advanced Master Mix for all TaqMan‑based detection methods.

We recommend using PowerTrack SYBR Green Master Mix for all SYBR Green‑based detection methods.

When using TaqMan Fast Advanced Master Mix (Cat. No. 4444965), how many times can I freeze-thaw my DNA samples?

The number of freeze-thaw cycles should be as low as possible to avoid the formation of ice crystals that can mechanically destroy the DNA. The maximum number of freeze-thaw cycles we recommend is 5. If you are using the same template repeatedly, we recommend preparing aliquots from a stock solution to avoid repeated freeze-thaw cycles.

What buffer should I store my DNA samples in?

DNA is most stable in slightly alkaline conditions (pH 7.5-8.0). Therefore, we recommend storing DNA samples in TE buffer which has a fixed pH of 8.0 and contains EDTA that binds any potential free radicals that can damage DNA.

I am using the TaqMan Gene Expression Cells-to-CT kit. Can I use the TaqMan Fast Advanced Master Mix in place of the TaqMan Gene Expression Master Mix to set up the qPCR reaction?

Yes, the TaqMan Fast Advanced Master Mix can be used in place of the TaqMan Gene Expression Master Mix when setting up the qPCR reaction for the TaqMan Gene Expression Cells-to-CT kit.

Can I use TaqMan Gene Expression Master Mix or TaqMan Universal Master Mix for TaqMan Advanced miRNA Assay?

TaqMan Advanced miRNA Assay was validated with TaqMan Fast Advanced Master Mix (Cat. No, 4444557) and our claims are based on the results with it. There is no technical reason why TaqMan Gene Expression Master Mix and TaqMan Universal Master Mix wouldn't work. They may be slightly less sensitive and will take a longer time to run.

Which master mix do you recommend for TaqMan Advanced miRNA Assay?

We recommend using TaqMan Fast Advanced Master Mix, Cat. No. 4444557.

For Research Use Only. Not for use in diagnostic procedures.