Karen L. Reece、Katharine Driftmier Miller Hoffmann、Cesar Corona、Thomas A. Kirkland、H. Tetsuo Uyeda、Stephen J. Dwight、Mark G. McDougall and Douglas R. Storts
Promega Corporation, Madison, Wisconsin, and Promega Biosciences, LLC., San Luis Obispo, Calif.
Publication Date: 2009
The new GoTaq® qPCR Master Mix is a 2X master mix for quantitative PCR (qPCR) featuring GoTaq® Hot Start Polymerase, aTuckerThe DNA polymerase is linked to a proprietary antibody that blocks polymerase activity at room temperature. Master Mix contains an optimized buffer formulation containing dNTPs and MgCl2And includes a new proprietary dsDNA-binding dye that can be used at higher concentrations than SYBR® Green I because it is less inhibitory in amplification reactions. The dye concentration in the master mix is optimized to produce significantly brighter fluorescence during qPCR than master mixes containing SYBR® Green I. Dye excitation and emission are similar to SYBR® Green I, so it is compatible with commonly used real mix.time PCR instruments.
GoTaq® qPCR Master Mix
Quantitative real-time PCR is a powerful tool for the detection and quantification of nucleic acids. By incorporating fluorescently labeled probes or nucleic acid or fluorescent double-stranded DNA (dsDNA)-binding dyes into PCR, product formation can be monitored after each cycle of PCR. Using real-time PCR has many advantages over endpoint PCR. Using fluorescent dyes to monitor amplification eliminates the need for gel electrophoresis, saving users time and reducing the possibility of contamination. Since product formation can be detected during the exponential phase of PCR, when reaction efficiency is highest, quantification of starting material is more precise and sensitive (1). The sensitivity offered by fluorochromes also allows detection over a wider dynamic range than endpoint PCR.
Probe-based chemistry, such as TaqMan® technology, is advantageous because labeled probes are amplicon-specific, producing a fluorescent signal during enzymatic processing of the probe:amplicon hybrid, indicating amplification Subspecific synthesis. However, these assays are difficult to design and more expensive than dye-based chemicals. dsDNA-binding dyes, such as SYBR® Green I, detect all dsDNA formed during amplification; however, these assay designs are simple and cost-effective. The ability to perform melting curve analysis (something not possible with TaqMan® assays) provides a quality control step to verify amplification of desired amplicons.
Master mix GoTaq® qPCR (number of cats. A6001) introduces a new proprietary dsDNA-binding dye, Bryt™ Green dye, which can be used at higher concentrations than SYBR® Green I due to its less inhibitory effect in amplification reactions. The dye concentration in the master mix is optimized to produce significantly brighter fluorescence during qPCR than a master mix containing SYBR® Green I. Dye excitation and emission are similar to SYBR® Green I (Figure 1), making it compatible with commonly used reagents and available instrument platforms. GoTaq® qPCR Master Mix is a 2X master mix consisting of an optimized buffer formulation with dNTPs and MgCl2 and featuring GoTaq® Hot Start Polymerase. GoTaq® Hot Start Polymerase consists of full-length Taq DNA polymerase conjugated to a proprietary antibody that prevents polymerase activity at room temperature. Heat activation is achieved by incubating the assembled reaction at 95 °C for 2 min. Proprietary polymerase/buffer formulations accommodate extended cycle numbers (45-50 cycles) and are compatible with thermal cycling programs including extended activation (95°C for 10 minutes). The Master Mix is premixed with a low concentration of CXR reference stain, identical to the ROX™ reference stain. The master mix is compatible with existing experimental designs and requires only the addition of DNA template, target-specific primers, and water.
"GoTaq® qPCR Master Mix features a new proprietary dsDNA-binding dye that can be used at higher concentrations than SYBR® Green I because it is less inhibitory in amplification reactions and produces significantly brighter fluorescence .The excitation and emission of the dye are similar to those of SYBR® GreenI".
Unless otherwise stated, 50 µL reactions were prepared and performed on an Applied Biosystems 7500 real-time PCR instrument in 9600 emulation mode and calibrated using the Pure Calibration Kit.Dye (Applied Biosystems) according to the manufacturer's instructions. Unless otherwise stated, cycling conditions were as follows: activation at 95°C for 2 minutes, denaturation at 95°C for 15 seconds, annealing/extension at 60°C for 60 seconds, 40 cycles, followed by 60°C to 95°C.
Brightness and Sensitivity
The brighter fluorescent signal provided by GoTaq® qPCR Master Mix allows earlier detection of amplification products compared to SYBR® Green I, resulting in earlier cycle of quantitation (Cq) values for many targets (Figure 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was demonstrated by amplifying serially diluted human genomic DNA (Cat.# G3041) using GoTaq® qPCR Master Mix or company A master mix in the same 96-well plate, demonstrating this feature. The instrument was programmed for an initial enzyme activation step of 10 min at 95°C to accommodate Company A's master mix requirements. The significantly brighter fluorescent signal produced by GoTaq® qPCR Master Mix allows software analysis to call Cq values up to three cycles earlier.
Note: Cq is often referred to as cycle threshold or CT. See Bustin, S. et al. (2009) Recommended qPCR terminology (2).
Amplification of the kanamycin phosphotransferase target sequence from plasmid DNA (Figure 3) showed efficient 8-order linear amplification with accurate quantification over the entire dynamic range. Alpha V was amplified from serial dilutions of cDNA prepared from a total RNA positive control (p/n C199A) and ImProm-II™ Reverse Transcriptase (cat # A3802). Figure 4 shows the specific and sensitive detection of one copy per reaction.
GoTaq® qPCR Master Mix is compatible with a variety of real-time PCR instrument platforms, using SYBR® Green I or preset filters for FAM™ and ROX™ as passive references. However, some instruments require a higher concentration of reference dye in the reaction mixture, depending on the instrument's detection settings. One tube of 100X CXR Reference Dye is supplied with the master mix. Below we list tested real-time PCR instruments and indicate which instruments require additional reference dyes.
GoTaq® qPCR Master Mix is compatible with a wide range of real-time PCR instruments
Instruments that do not require the addition of 100X CXR Reference Dye:
- Applied Biosystems 7500 and 7500 Fast Real-Time PCR Systems
- Stratagene Mx3005P® Quantitative PCR System
- Roche LightCycler® 480
- BioRad Chromo4™ Real-Time Detector
- Eppendorf Mastercycler® ep realplex4 y realplex4 S
Instruments that require addition of 100X CXR Reference Dye at 1X per reaction:
- Applied Biosystems 7000 Sequence Detection System
- Applied Biosystems 7300 Real-Time PCR System
- Applied Biosystems 7700 Sequence Detection System
- Applied Biosystems 7900HT Real-Time PCR System
- Applied Biosystems StepOne® and StepOne® Plus Real-Time PCR Systems
Bio-Rad real-time instruments such as the iCycler®, iQ™5, and MyCycler™ perform normalization by collecting dynamic well factors using the background signal detected in the first few PCR cycles. Because SYBR® Green I uses insufficient background for the instrument to perform these calculations, the Bio-Rad protocol requires the addition of 10 nM fluorescein to each reaction (3). GoTaq® qPCR Master Mix was run on a Bio-Rad iQ™5 instrument with and without fluorescein. Figure 5 shows that amplification of GAPDH from serially diluted human genomic DNA was linear and efficient with or without the addition of fluorescein to the master mix. This demonstrates that the brightness of Promega's proprietary dye provides sufficient signal for the instrument to collect dynamic pore factors without the need for the addition of fluorescein. When using a Bio-Rad instrument with GoTaq® qPCR Master Mix, users must run test reactions with their experimental objectives to determine if fluorescein needs to be added to the master mix.
Many companies offer individual master mixes containing enzymes compatible with fast activation times and faster cycling conditions. GoTaq® qPCR Master Mix is compatible with fast cycling protocols (Figure 6). Assembly reactions (20 µl) using GAPDH-specific primers and serial dilutions of human genomic DNA. GoTaq® qPCR Master Mix and Company A Rapid Master Mix were run on separate plates on an Applied Biosystem 7500 FAST Real-Time PCR System using predetermined fast cycling conditions (20 sec activation at 95°C, 40 cycles). Denaturation at 95 °C for 3 s and annealing/extension at 60 °C for 30 s, melting curve analysis from 60 °C to 95 °C). GoTaq® qPCR Master Mix performed similarly to Company A's Rapid Master Mix and showed a significantly brighter fluorescent signal.
GoTaq® qPCR Master Mix provides superior amplification and fluorescence quality in a convenient 2X formulation that includes a new proprietary dsDNA-binding dye that produces a significantly brighter signal than SYBR® Green I during qPCR. Since the spectral properties of this colorant are similar to SYBR® Green I, the premix can be easily replaced with other color-based premixes
qPCR Master Mix. The convenient 2X Master Mix only needs to add experimental template DNA, primers and water. It is compatible with common thermal and rapid cycling protocols and can be used with any real-time instrument capable of detecting SYBR® Green I or FAM™ dyes.
- Sandquist (2005)The key to real-time quantitative PCR.Promega eNotes website.
- Bustin, S.A.等。 (2009)MIQE Guidelines: Minimum Information for Publishing Quantitative Real-Time PCR Experiments.clinical. Chemistry 55, 611–22.
- BioRad iCycler iQ™ Real-Time PCR Detection System Instruction Manual, 4006200 Rev E.
GoTaq® Green Master Mix contains two dyes (blue and yellow) that allow monitoring of progress during electrophoresis. Reactions assembled with GoTaq® Green Master Mix have sufficient density for direct loading onto agarose gels.What are the components of the GoTaq master mix? ›
Both Master Mixes are ready-to-use 2X solutions that contain GoTaq® DNA Polymerase, dNTPs, MgCl2 and reaction buffer at optimal concentrations for efficient amplification of DNA templates by PCR. GoTaq® Green Master Mix also includes two dyes (blue and yellow) that allow monitoring of progress during electrophoresis.What is the shelf life of GoTaq Green Master Mix? ›
Product may be stored at 4°C for up to 6 weeks. Mix well prior to use. Functional Assay: GoTaq® Green Master Mix is tested for performance in the polymerase chain reaction (PCR). GoTaq® Green Master Mix, 1X, is used to amplify a 360bp region of the α-1-antitrypsin gene from 100 molecules of human genomic DNA.What is the green dye for qPCR? ›
SYBR® Green for qPCR
Sso7d is a double-stranded (ds) DNA–binding protein that increases speed and processivity and increases tolerance to PCR inhibitors. SYBR® Green is a dsDNA-binding dye that intercalates nonspecifically into dsDNA, allowing measurement of the amount of PCR product.
The GoTaq® G2 Hot Start Colorless Master Mix contains no gel loading dye for use when downstream applications require fluorescence or absorbance readings without purification. Performance Guarantee: Promega PCR systems, enzymes and reagents are proven in PCR to ensure reliable, high-performance results.What is the composition of SYBR Green Master Mix? ›
The 1 x 5 mL vials contain a 2X mixture of SYBR Green 1 Dye, AmpliTaq Gold™ DNA Polymerase, dNTPs with dUTP, Passive Reference 1 (ROX), and optimized buffer components. Sufficient reagents provided for 200 reactions based on a 50 μl reaction volume.What is the GoTaq master mix for qPCR? ›
The GoTaq® qPCR Master Mix is a ready-to-use 2X master mix for real-time quantitative PCR. Combining GoTaq® Hot Start Polymerase, optimized buffer and the BRYT Green® Dye, the GoTaq® qPCR Master Mix provides robust real-time PCR with earlier quantification cycle values and broad-range detection.What is the purpose of master mix in PCR? ›
The master mix enables researchers to set up controls and test different concentrations of their target DNA or RNA templates without having to individually add precise amounts of enzymes, buffers, cofactor (usually MgCl2), water and dNTP to each reaction tube or plate well.What is the master mix and why do you need each component? ›
What is the master mix and why do you need each component? It contains all the components for PCR mix to occur; including the individual building blocks of DNA (nucleotides, or dNTP's), a special buffer to maintain optimum pH, salts, and MgCl2.How do you store PCR master mix? ›
- Long term storage at -20 °C. Product expiry at -20 °C is stated on the label.
- Option: Store at +4 °C for up to 6 months.
- RealQ Plus master mixes can be kept at + 4°C for up to 3 months.
Aliquots of master mix may be stored at -20 C, but repeated freezing and thawing will affect the efficiency of Taq polymerase. You definitely can do this but storing it for long time could cause some problems .What are the steps of PCR? ›
PCR is based on three simple steps required for any DNA synthesis reaction: (1) denaturation of the template into single strands; (2) annealing of primers to each original strand for new strand synthesis; and (3) extension of the new DNA strands from the primers.What is dye based qPCR? ›
Dye-based quantitative PCR (qPCR) uses real-time fluorescence of a double-stranded DNA (dsDNA) binding dye (e.g., SYBR® Green) to measure DNA amplification as it occurs during PCR. The dye displays weak background fluorescence that increases dramatically upon binding to dsDNA.What is the difference between TB green and SYBR Green? ›
The TB Green Advantage qPCR Premix demonstrated superior reaction specificity when compared to Competitor I's SYBR qPCR master mix. This was indicated by tighter peaks in the melting curve for the Takara Bio enzyme.What is the difference between SYBR Green and EVA green? ›
Dye stability: EvaGreen® dye is very stable both during storage and under PCR conditions. SYBR® Green I, on the other hand, is known to degrade following multiple freeze-thaw cycles and under PCR conditions. Moreover, decomposed SYBR® Green I is reported to be even more inhibitory to PCR.What is Mastermix primer? ›
The mastermix is suitable to amplify any eubacterial DNA. The amplified region (approx. 450 bp) contains variable sequences for the identification of bacteria by taxon specific probing or sequence analysis. Mastermix 16S Primer is a 2.5x-concentrated solution, the final volume of the reaction mixture being 25 µl.What is OneTaq hot start 2X master mix from Neb? ›
- Obtain high yields across a wide range of AT / GC content.
- 2X higher fidelity than Taq.
- Hot Start feature enables room temperature reaction setup.
- Master mix is a 2X concentrated solution containing everything needed for robust amplification.
GoTaq® Hot Start Master Mixes are premixed, ready-to-use solutions containing GoTaq® Hot Start Polymerase, magnesium, dNTPs and buffer. Reactions can be set up in less than a minute at room temperature; simply add your template, water and primers.What is the disadvantage of SYBR Green dye? ›
Disadvantage of SYBR dye
The primary disadvantage is that it may generate false positive signals; i.e., because the SYBR dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences.
The most important difference between the TaqMan and SYBR Green I dye chemistries is that the SYBR Green I dye chemistry will detect all double-stranded DNA, including non-specific reaction products. A well-optimized reaction is therefore essential for accurate results.
Furthermore, SYBR Green has a medium specificity while Taqman has high specificity. Moreover, the reproducibility of SYBR Green is also medium while the reproducibility of Taqman is high. SYBR Green and Taqman are two chemistries used to detect PCR products in real-time PCR procedures.Why use a PCR Mastermix rather than adding individual reagents? ›
Using a master mix has several advantages: First, the number of single pipetting steps is reduced. In this way, both the risk of user errors during pipetting and the risk of contamination are minimized and, of course, time is saved. In principle, the pipetting accuracy is also higher, since larger volumes are dosed.What are the 3 components of PCR master mix? ›
Components. A PCR master mix consists of a thermostable DNA polymerase, optimized reaction buffers, dNTPs, and MgCl2 (or often MgSO4).Can I use PCR master mix for qPCR? ›
PCR Master Mixes and Supermixes
Only template, primers, probes (if being used), and water, to make up the volume, need to be added. One or more dyes may be included for fluorescent detection of product formation in real-time quantitative PCR (qPCR).
A master mix enables faster setup with less pipetting as the mix can be prepared at one time and divided among multiple pipettes to save time. Researchers don't have to spend time individually adding precise amounts of buffers, enzymes, dNTP, cofactor and water to each reaction tube or plate well.What is the importance of creating a master mix PCR lab quizlet? ›
What is a PCR master mix and what is the benefit of making it? **The master mix contains a thermostable DNA polymerase, gene-specific forward and backwards primers, each of the 4 dNTPs, reaction buffer and MgCl2. **The mix will drive the PCR, increase efficiency, and decrease the risk of contamination.What should you do after knowing the master mix volume? ›
Once your master mix is finished, well mixed and dispensed into tubes or plates, you can add the template DNA. As the DNA samples are usually highly viscous and needed in small quantities, you should either dispense them into the master mix or onto the wall of the tube or well.Why shouldn t you mix your DNA template to the master mix you have prepared? ›
Once you've prepared your master mix and pipetted it into the PCR tubes, stop. Don't add the DNA template until the instuctor tells you, because we're all going into the same PCR machine and need to start at the same time.What is the meaning of master mix? ›
A master mix is a mixture containing precursors and enzymes used as an ingredient in RT-PCR techniques in molecular biology.How long can I keep the prepared master mix before running the real-time PCR reaction? ›
The stability of PrimeTime Gene Expression Master Mix makes it ideal for overnight PCR runs and high throughput experiments using robotic liquid handling systems. Reliable results have been obtained when the qPCR was run directly after setup or after the reactions were maintained at room temperature for up to 72 hr.
Answer and Explanation: When conducting a PCR and making the PCR master mix, RNA is not added to the sample. DNA is added that is going to be amplified. DNA nucleotides and a heat stable DNA polymerase are necessary to polymerize and amplify the target DNA.How long does PCR master mix last? ›
PCR Master Mix allows you to set up your reactions in less than a minute—just add template and primers. Optimized conditions enable amplification of as few as 2 copies of target template. PCR Master Mix is stable for 3 months when stored at 4°C.What is the ratio of master mix to PCR? ›
The Genaxxon bioscience PCR Mastermix (2X) is provided as a 2X concentrated (i.e. a 12.5µL volume of PCR Mastermix (2X) is required for PCR reactions with a final volume of 25µL). For volumes smaller than 50µL, the 1:1 ratio of PCR Mastermix (2X) to diluted primer mix, template DNA and water should be maintained.Can I prepare a qPCR plate in advance? ›
Typically, we recommend running RT-qPCR reactions as soon as possible after reactions are set up.At what temperature is Taq polymerase deactivated? ›
As expected, Taq DNA polymerase is inactive at low temperatures below 30 °C and its activity continues to increase at up to 72 °C.What temperature is TaqMan? ›
TaqMan assay in solution
The amplification condition consists of an initial 2 min at 50°C for optimizing the UNG enzyme, and 10 min denaturation at 95°C followed by 40 cycles of 30 s of denaturing at 95°C, 30 s of annealing at 52°C, and 60 s of extension at 65°C.
Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied.What are the three temperatures of PCR? ›
|Standard 3-step PCR Cycling|
|Cycle step||Temperature||Number of Cycles|
|Initial Denaturation||94 °C to 98 °C||1|
|Denaturation Annealing Extension||94 °C 5 °C below Tm 70 °C to 80 °C||25-35|
|Final Extension||70 °C to 80 °C||1|
Colony PCR is a method for rapidly screening colonies of yeast or bacteria that have grown up on selective media following a transformation step, to verify that the desired genetic construct is present, or to amplify a portion of the construct.What is green taq? ›
Green Taq DNA Polymerase is designed to increase the stability of the Taq enzyme for more convenient transport and applications. The polymerase (1000 U) is designed for 400 rxns if 2.5 U are used per 50 ul PCR reaction.
The master mix usually includes DNA polymerase, dNTPs, MgCl2 and buffer. Using a master mix reduces pipetting and risk of contamination, is convenient, saves time and preempts possible errors in mixing, making it ideal for high-throughput applications.What are the advantages of PCR master mix? ›
Benefits of PCR Mix
With a PCR master mix, you can get a faster setup with fewer pipettes. Once a mix is prepared, it can be divided among pipettes to save time. Reduced scope for pipetting reduces experimental error, contamination, and variability between tubes.
There are 3 strategies for primer design: 1) insert-specific primers, 2) backbone-specific primers, and 3) orientation-specific primers.What can cause a colony PCR to fail? ›
These include, but are not limited to, agar from bacterial plates, high concentrations of DNA / bacterial debris, or incidental contamination of PCR solutions with the original backbone vector. Insufficient DNA or suboptimal thermocycling programs can also produce false negatives, but at lower rates than contamination.Is SYBR Green cheaper than TaqMan? ›
Compared to the SYBR Green assay, the use of TaqMan probes is more expensive, but also offers two significant advantages: the TaqMan assay only measures amplification progression of the target sequence, as the probes are target specific.What is the role of SYBR Green in PCR? ›
SYBR Green is one of the most commonly used fluorescent dyes in qPCR. It binds to double-stranded DNA molecules by intercalating between the DNA bases. Once intercalated to DNA, SYBR Green becomes less mobile, causing its energy to be released as fluorescence.How does SYBR Green work in PCR? ›
SYBR® Green chemistry is a method for performing real-time PCR analysis. SYBR Green dye binds the minor groove of double-stranded DNA. When SYBR Green dye binds to double-stranded DNA, the intensity of the fluorescence increases. As more double-stranded amplicons are produced, SYBR Green dye fluorescence increases.What are the 4 steps of PCR test? ›
The PCR process has 4 steps:collection, preparation, amplification, and post PCR clean-up. The PCR machine steps happen in the amplification step.What is in the master mix for PCR? ›
PCR Master Mix is a premixed, ready-to-use solution containing Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR.