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View additional product information for TaqMan™ Fast Advanced Master Mix for qPCR - FAQs (4444557, 4444964, 4444556, 4444963, 4444965, 4444558)
31 product FAQs found
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)
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).
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).
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.
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.
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.
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).
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.
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).
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.
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.
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.
No. A TaqMan probe, once cleaved, cannot be re-quenched. Therefore a melt curve does not apply when using a TaqMan assay.
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.
Please view this short video (https://www.youtube.com/watch?v=eIaPGhOjBQo), which explains some best practices for replicates and plate setup.
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.
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 two-step. 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 gives greater flexibility. This table (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/1-step-vs-2-step.html) also provides a comparison.
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.
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.
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.
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.
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.
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.
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.
All materials required for TaqMan® Advanced miRNA Assay workflow using TaqMan® Advanced miRNA Human and B cards (Cat. A31805) can be found on pages 9-11, in the TaqMan® Advanced miRNA Assays User Guide.
Reagents and kits required for the TaqMan® Advanced miRNA Assay workflow include, but are not limited to:
• RNA Isolation Kit (see recommended kits on page 9)
• TaqMan® Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007)
• TaqMan® Fast Advanced Master Mix (Cat. No. 4444557)
Please refer to the user guide linked above for a full list of other materials and equipment required for the workflow.
Find additional tips, troubleshooting help, and resources within our Epigenetics Support Center.
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.
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.
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.
Yes, the TaqMan Fast Advanced Master Mix (Cat. No. 4444557) can be used in place of the TaqMan Gene Expression Master Mix (Cat. No. 4369016) when setting up the qPCR reaction for the TaqMan Gene Expression Cells-to-CT kit.
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.
We recommend using TaqMan Fast Advanced Master Mix, Cat. No. 4444557.