QuantiFast SYBR® Green RT-PCR Kit

For fast, one-step qRT-PCR using SYBR Green I for gene expression analysis

Products

The QuantiFast SYBR® Green RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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QuantiFast SYBR Green RT-PCR Kit (2000)

Cat. No. / ID:  204156

IMPORTANT NOTE: As announced earlier, the production of the QuantiFast kits has been discontinued since mid-2021. Hence, these products will be available only until stocks last.   For 2000 x 25 µl reactions: 25 ml 2x QuantiFast SYBR Green RT-PCR Master Mix (contains ROX dye), 0.5 ml QuantiFast RT Mix, 20 ml RNase-Free Water
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4.660,00 $
This kit is being phased out. We recommend switching to the QuantiNova successor product. For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.

Features

  • Faster results with time savings of up to 60%
  • Sensitive detection of even low copy numbers
  • Accurate detection of a wide range of template amounts
  • Optimized, ready-to-use master mix for fast cycling
  • Universal protocol for all standard and fast cyclers

Product Details

The QuantiFast SYBR Green RT-PCR Kit delivers fast and specific quantification of RNA targets by real-time one-step RT-PCR using SYBR Green I detection. Q-bond technology and an optimized, ready to use master mix enable shorter real-time RT-PCR run times, not only on fast cyclers with short ramping times, but also on standard cyclers. The combination of a hot start and a unique qRT-PCR buffer system ensures highly specific and sensitive real-time quantification of RNA targets. The QuantiFast SYBR Green RT-PCR Kit is also supplied with an optimized RT mix for efficient cDNA synthesis in only 10 minutes. For convenience, the master mix can be stored at 2–8°C.

IMPORTANT NOTE: As announced earlier, the production of the QuantiFast kits has been discontinued since mid-2021. Hence, these products will be available only until stocks last. Visit the product page of the successor kit to view improved features or to request a trial kit.

For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.

Performance

The QuantiFast SYBR Green RT-PCR Kit delivers highly specific and sensitive results, outperforming other real-time RT-PCR kits used in fast cycling mode (see figure " Specific one-step RT-PCR"). RT-PCR run times are reduced by up to 60% (see figure " Significantly reduced PCR times"), allowing you to get results faster. You can also greatly increase your sample throughput or efficiently share a cycler with other users.

Results in one-step RT-PCR are comparable to those achieved in two-step RT-PCR (see table).

Comparable performance in two-step and one-step RT-PCR on the Applied Biosystems 7500 Fast System
Mean CT value
Amount of cDNA/RNA QuantiFast SYBR Green PCR Kit (two-step RT-PCR) QuantiFast SYBR Green RT-PCR Kit (one-step RT-PCR)
10 ng 20.27 20.11
1 ng 23.44 23.46
0.1 ng 27.18 26.86
NTC 45.00 45.00

QuantiFast SYBR Green RT-PCR Kit enables accurate quantification of targets over several log dilutions of template (see figure " Detection over 7 logs of template"). Even small differences in the amount of low-copy targets can be clearly distinguished.

See figures

Principle

The QuantiFast SYBR Green RT-PCR Kit delivers highly sensitive and specific results over a wide dynamic range on both standard and fast cyclers. The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. A specially developed fast RT-PCR buffer contains the novel PCR additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure " Fast primer annealing"). A balanced combination of K+ and NH4+ ions in the PCR buffer promotes specific primer annealing, enabling high RT-PCR specificity and sensitivity (see figure " Specific primer annealing"). In addition, HotStarTaq Plus DNA Polymerase requires only 5 minutes at 95°C for activation and provides a stringent hot start, preventing the formation of nonspecific products. The optimized QuantiFast RT Mix enables cDNA synthesis in just 10 minutes. 

Components of 2x QuantiFast SYBR Green RT-PCR Kit*
Component Features and benefits Benefits
HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature
QuantiFast SYBR Green RT-PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
Unique Q-Bond additive Faster PCR run times, enabling faster results and more reactions per day
SYBR Green I dye Yields a strong fluorescent signal upon binding to double-stranded DNA Highly sensitive quantification
ROX dye Normalizes fluorescent signals on Applied Biosystems and, optionally, Agilent instruments Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler
QuantiFast RT Mix Special blend of reverse transcriptases with high affinity for RNA RNA can be transcribed in just 10 minutes, even through complex secondary structures
See figures

Procedure

The QuantiFast SYBR Green RT-PCR Kit is a ready-to-use master mix that eliminates the need for optimization of reaction and cycling conditions. Simply add primers and template to the ready-to-use PCR master mix, and start the reaction. Follow the protocol in the handbook to get fast and reliable results on any real-time cycler.

For optimal results in real-time, one-step RT-PCR, we recommend using the QuantiFast SYBR Green RT-PCR Kit in combination with QuantiTect Primer Assays. They are bioinformatically validated primer sets for any gene from human, mouse, rat, and many other species. Assays can be easily ordered online at the GeneGlobe Web portal.

Applications

The QuantiFast SYBR Green RT-PCR Kit is for use in gene expression analysis of RNA targets. QuantiFast SYBR Green RT-PCR Kits are compatible with all available real-time cyclers, including instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene SYBR Green RT-PCR Kit, which has been specially developed for fast cycling on these instruments.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsSYBR Green-based, real-time, one-step RT-PCR
Single or multiplexSingle
Real-time or endpointReal-time
SYBR Green I or sequence-specific probesSYBR Green I
Reaction typeReal-time and one-step RT-PCR
Thermal cyclerAll real-time cyclers (e.g. LC, RG, ABI)
Sample/target typeRNA
With or without ROXWith ROX

Resources

Kit Handbooks (2)
For fast, quantitative, real-time, one-step RT-PCR using SYBR Green I
For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
Certificates of Analysis (1)

FAQ

Does the high primer concentration required by QuantiFast SYBR Green PCR Kits impair annealing specificity?

No, high annealing specificity with the QuantiFast SYBR Green PCR Kits is maintained due to an intrinsic hot-start and the buffer composition.

 

FAQ ID -1444
How do I setup and validate a multiplex PCR assay with QIAGEN PCR kits?

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

 

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

 

FAQ ID -9093
Do the QuantiTect Primer Assays work with QuantiFast SYBR Green Kits?

We have performed numerous tests comparing the performance of QuantiFast Kits and QuantiTect Kits with QuantiTect Primer Assays. Due to the optimized ion concentrations in the PCR buffers, both perform equally well with QuantiTect Primer Assays and do not require any adjustment of primer concentration.

 

FAQ ID -1439
Can QuantiFast PCR Kits be used on real-time PCR instruments without fast cycling options?

Yes, QuantiFast Kits can also be run on a qPCR cycler without fast cycling options. You cannot achieve rapid ramping rates, but you can still take advantage of the combined annealing/extension step and the reduced denaturation and annealing/extension times offered by QuantiFast Kits.

You will be able to obtain your PCR results in a much shorter time.

 

FAQ ID -1428
Is the master mix of QuantiFast Kits for real-time PCR aliquoted into several tubes to prevent cross-contamination?

QuantiFast Kits for 400 x 25 µl reactions contain a master mix that is aliquoted into 3 separate tubes.

QuantiFast Kits for 2000 x 25 µl reactions provide one tube containing 25 ml master mix to offer a cost-effective solution for higher throughput experiments.

 

 

FAQ ID -1697
Do you have any information or guidelines regarding the choice of reference genes for real-time PCR?

Yes, please visit our website section 'Using endogenous control genes in real-time RT-PCR' for general information. It provides a list of relative gene expression levels for commonly used human and mouse reference genes.

We offer a set of ready-to-order control genes for use in SYBR Green based as well as probe based real-time RT-PCR.

In addition, you may want to refer to the following citations on reference gene selection for quantitative real-time PCR:

• Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, DePaepe A, Speleman F [2002]: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:0034.

• Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A., 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313(4): 856-62.

• Katrien Smits,Karen Goossens, Ann Van Soom, Jan Govaere, Maarten Hoogewijs, Emilie Vanhaesebrouck,Cesare Galli, Silvia Colleoni, Jo Vandesompele, and Luc Peelman [2009]Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes. Dec 11;2:246.

FAQ ID -2371
Why is the reaction volume for QuantiFast PCR Kits lower than that for QuantiTect PCR Kits?

The reduced reaction volume recommended for QuantiFast PCR Kits compared to QuantiTect PCR Kits allows more efficient temperature transfer during short cycling steps.

 

FAQ ID -1447
Do you offer trial-kit sizes for the new QuantiFast Kits?

Yes, the QuantiFast SYBR Green PCR Kit and QuantiFast Probe PCR Kits are available for 80 x 25 µl reactions. This trial-kit size is not available for QuantiFast RT-PCR Kits.

 

FAQ ID -1429
Why do replicates in real-time PCR have different plateau heights?

Replicates in real-time PCR may have different plateau heights due to differences in the reaction kinetics for each sample. Even though replicates start out with identical template amounts, the rate at which reagents are being depleted, and the point when exponential accumulation of PCR product stops and becomes linear, differ between replicates. This will result in different plateau heights, the stage where PCR reactions have come to a halt, and little or no additional PCR product is being amplified. You can find further information in Chapter 'Quantification of target amounts' of our Brochure "Critical Factors for Successful Real-Time PCR".

 

FAQ ID -539
Will QuantiTect Primer Assays work at an annealing temperature of 60ºC with QuantiFast SYBR Green PCR and RT-PCR Kits?

Yes. QuantiTect Primer Assays are guaranteed for use with QuantiFast SYBR Green PCR and QuantiFast SYBR Green RT-PCR Kits at the specified annealing temperature of 60ºC. We have extensively tested many QuantiTect Primer Assays with success under these conditions.

 

FAQ ID -1553
How do I create a workspace that is free of DNA contamination, prior to carrying out a qPCR experiment?

Any DNA contamination will artificially inflate the SYBR Green signal, yielding skewed gene expression profiles and false-positive signals. The most common source of DNA contamination is from PCR products generated during previous experiments. Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers. To minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:

 

  • Remove a single aliquot of water from your PCR-grade stock, sufficient to complete the experiment. This minimizes the number of times that the stock container is opened, thereby minimizing contamination risks.
  • Use only fresh PCR-grade reagents and disposable labware.
  • Treat any labware (tubes, tips, and tip boxes) used in PCR with 10% bleach, before discarding.
  • Maintain a dedicated workspace for PCR setup (perhaps a PCR-only hood), away from areas of the lab where post-PCR work is done, such as running gels, enzyme digestions, and cloning.
  • Change the lab bench pads/papers often and decontaminate lab benches and labware (racks, pipettors, etc.) before each use by washing with 10% bleach, and/or exposing to UV light for at least 10 minutes. This serves to degrade and/or inactivate contaminating DNA.
  • Before, during, and after the experiment, minimize the opening and closing of any tubes or plates used during the experiment.  
FAQ ID -2654
Do you have information on the use of recombinant DNA and RNA as absolute standards for realtime RT-PCR?

Recombinant DNA (recDNA) is very stable and represents the average size of mRNA. Due to the cloning and purification processes, obtaining recDNA can lengthen the overall process of generating standards.

Recombinant RNA (recRNA) and native RNA undergo reverse transcription as well as PCR, and mimic the natural process for mRNA in RT-PCR. Complicated cloning and purification of recRNA and instability of recRNA are two disadvantages for using recRNA as a standard. For further details please refer to the section "Generating Standard Curves" in Appendix D of the QuantiTect SYBR Green PCR Handbook.

FAQ ID -729
What is a QuantiTect Primer Assay?

QuantiTect Primer Assays are primer pairs designed and bioinformatically validated specifically for real-time RT-PCR with SYBR Green detection. To find a primer assay for your target gene of interest, please visit our GeneGlobe data base.

For best results, we strongly recommend using QuantiTect Primer Assays in combination with QIAGEN's products for SYBR Green-based Real-Time PCR and RT-PCR.

FAQ ID -1141
How many reactions can I perform with the new QuantiFast Kits for real-time PCR?

Compared with QuantiTect Kits, the recommended reaction volume for QuantiFast Kits is reduced from 50 µl to 25 µl (96-well block cyclers), and from 20 µl to 10 µl (384-well block cyclers).

The volume of master mix remains the same, which means that QuantiFast Kits offer twice the number of reactions as QuantiTect Kits. However, for LightCycler instruments, the recommended reaction volume remains the same (20 µl).

FAQ ID -1425
Can I use uracil-N-glycosylase (UNG) with the QuantiFast and Rotor-Gene PCR kits?

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092
What annealing temperature should be used with the QuantiTect Primer Assays?

The annealing temperature for QuantiTect Primer Assays should be 55oC when used with the QuantiTect SYBR Green PCR Kit and the QuantiTect SYBR Green RT-PCR Kit.

Use of the QuantiFast SYBR Green PCR and QuantiFast SYBR Green RT-PCR Kits requires a combined annealing/extension step at 60oC, as described in the QuantiTect Primer Assay Handbook.

Note that these Assays are guaranteed for use with the QuantiTect or QuantiFast chemistries only!

 

 

FAQ ID -849
Why do QuantiFast SYBR Green PCR Kits require such a high primer concentration?

The high primer concentration (1 µM) required for the QuantiFast SYBR Green PCR Kits allows efficient hybridization during the shortened annealing time. This high concentration is well suited for both one-step and two-step RT-PCR.

 

FAQ ID -1443
Why is the RT step with the QuantiFast RT Kits much shorter compared to QuantiTect RT Kits?

The combination of Omniscript and Sensiscript Reverse Transcriptases was optimized in the QuantiFast RT-PCR Kits. In addition, an optimized dNTP concentration and the limitation of amplicon size to <300 bp allow to reduce the time for the reverse transcription step to only 10 minutes.

 

FAQ ID -1451
Why is the storage time for QuantiFast PCR Kits shorter than that for QuantiTect PCR Kits?

The storage time for QuantiFast PCR Kits is shorter than for QuantiTect PCR Kits, because all QuantiFast master mixes contain HotStarTaq Plus DNA Polymerase, instead of HotStarTaq DNA Polymerase which requires longer activation times.

Excessive exposure to elevated temperatures will result in reactivation of the HotStarTaq Plus DNA Polymerase, eventually leading to nonspecific amplification.

 

FAQ ID -1446
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70ºC?

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

 

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

 

FAQ ID -90990
How do I quantify gene expression levels if the amplification efficiencies are different between the genes of interest and endogenous reference gene?

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095
Have QuantiTect Primer Assays been tested with QuantiFast SYBR Green PCR Kits on the Mastercycler ep realplex?

Yes, QuantiTect Primer Assays work very well under fast-cycling conditions. Results achieved with QuantiFast SYBR Green Kits are comparable to those achieved with QuantiTect SYBR Green Kits.

 

FAQ ID -1714
How important is the RNA purification process, for obtaining reliable qRT-PCR results?

The most important prerequisite for any gene expression analysis experiment is the preparation of consistently high-quality RNA from every experimental sample. Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment.

Genomic DNA contamination in an RNA sample compromises the quality of gene expression analysis results. The contaminating DNA inflates the OD reading of the RNA concentration. It is also a source of false positive signals in RT-PCR experiments.

RNase contamination degrades RNA samples whichcauses low signal and false-negative results in PCR.

Residual polysaccharides, collagen, other macromolecules, and organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal. These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity.

For fast purification of high-quality RNA we recommend QIAGEN’s RNeasy Kits like the RNeasy Mini Kit, the RNeasy Plus Universal Kit, or the RNeasy FFPE Kit.

FAQ ID -2655
What is the threshold cycle or Ct value?
The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.
FAQ ID -2682
QuantiTect Primer Assays are bioinformatically validated, genomewide primer sets. What does “bioinformatically validated” mean?

For each QuantiTect Primer Assay, we retrieve the sequence of the target gene from curated databases and exclude SNP regions from assay design.

We then use our proprietary algorithm to design assays that amplify RNA sequences only (i.e., at least one primer overlaps a splice site), provided that information on the position of splice sites is available. The assays are designed to provide optimal performance with QuantiFast and QuantiTect SYBR Green Kits.

After assay design, we validate randomly selected QuantiTect Primer Assays using real-time RT-PCR to check their compatibility with various real-time cyclers. Both two-step and one-step RT-PCR are carried out. Successful amplification of the target is indicated by the following factors: high sensitivity, high efficiency, high specificity (i.e., a single peak in melting curve analysis), and no primer–dimers in the no-template control (NTC).

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

 

FAQ ID -1982
What do I do if no fluorescent signal is detected in a real-time PCR assay?

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

 

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line.  Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

 

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091
How do you achieve fast cycling, yet still deliver the same performance in PCR as that achieved with standard cycling?

Our unique multiplex PCR buffer system with ammonium and potassium ions and Factor MP has been further optimized in QuantiFast and Rotor-Gene Kits. We have also discovered Q-Bond, a buffer component which supports the rapid formation of the polymerase–primer–template complex, leading to reduced annealing times.

FAQ ID -1430
How do I select appropriate reporter and quencher combinations for multiplex PCR?

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

 

FAQ ID -9094
Can I skip the gDNA wipeout buffer treatment step for the QuantiTect Reverse Transcription Kit?

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098
Why does my realtime PCR assay quality decrease over time?
Make sure that template, primers, probes, and amplification reagents are stored correctly and avoid multiple freeze–thaw cycles for oligonucleotides and template. Check the performance of your real-time instrument as some instruments require the halogen lamp to be frequently replaced. Lasers must also be replaced occasionally.
FAQ ID -589
Do special settings have to be used for QuantiFast PCR Kits on the Eppendorf Mastercycler ep realplex?

No. Optimized thermal cycling programs for use with QuantiFast Kits and a Program Selection Guide are available.

To install these programs on your Mastercycler ep realplex, contact your Eppendorf sales representative or visit our QIAGEN/Eppendorf Alliance page.

 

FAQ ID -1437
How much time will be saved when switching from standard cycling to fast cycling with QuantiFast Kits?

Depending on the qPCR instrument, time savings when switching from standard cycling (e.g., QuantiTect PCR Kits) to fast cycling using QuantiFast Kits range from 40% to 60%.

 

 

FAQ ID -1438
How should I handle and store absolute quantitation standards for real-time experiments?
Store the standards at a high concentration in aliquots at -20oC to -70oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.
FAQ ID -9099
How do I ensure reliable results for High Resolution Melting (HRM) assays?

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

 

  • Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
  • DNA template concentrations should be normalized using the same dilution buffer. Ensure the CT values are below 30 and differ no more than 3 CT values across individual samples.
  • Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
  • Always start with 0.7 µM primer concentration

 

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097
What is Q-Bond used in the QIAGEN Fast Cycling PCR and QuantiFast Kits?

The Q-Bond Molecule, present in the PCR Buffer of the QIAGEN Fast Cycling PCR Kit and the QuantiFast Kits, dramatically increases the binding affinity of DNA polymerase to single-stranded DNA, thereby facilitating the reduction of annealing time to just a few seconds. It is a non-protein PCR component. Unfortunately, all further information on this molecule is proprietary.

FAQ ID -1554
Why is the activation time for HotStarTaq Plus Polymerase in the QuantiFast SYBR Green Kits different from that for QuantiFast Probe Kits?

The activation time for HotStarTaq Plus DNA Polymerase used in the QuantiFast SYBR Green PCR Kits is longer than that for QuantiFast Probe PCR Kits. This is due to differences in buffer composition. Buffer components such as salts and additives influence the time required for enzyme activation.

 

FAQ ID -1449
Why should DNA or cDNA targets be less than 250 bp long for real-time PCR?

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

FAQ ID-751
What should I use as a standard for absolute quantification in real-time PCR?

For quantification of RNA, we strongly recommend using RNA molecules as standards. Use of in vitro transcripts as standards takes into account the variable efficiency of the RT reaction. An alternative to the use of in vitro transcripts as RNA standards is the use of a defined RNA preparation (e.g., from a cell line or virus preparation), for which the absolute concentration of the target has already been determined.

For quantification of DNA, several types of DNA can be used, such as plasmids, PCR products, or genomic DNA.

For more information, see Appendix E 'Generating Standard Curves' in the QuantiTect Probe PCR Handbook.

FAQ ID -1085
Can 2 µl reaction volumes be used with QuantiFast PCR Kits?

We recommend a reaction volume of 10 µl when using 384-well blocks with QuantiFast PCR Kits. If reducing the reaction volume to 2 µl, results will vary depending on the real-time cycler used.

Please contact QIAGEN Technical Services for more information.

FAQ ID -1440
Why is the QuantiFast denaturation step different for PCR and RT-PCR runs in the two-step protocol for the ABI 7500 and other cyclers?

This is due to differences in composition between PCR and RT-PCR buffers. QuantiFast PCR Buffers are optimized for fast amplification with shortest possible PCR steps, while QuantiFast RT-PCR Buffers are optimized for reverse transcription and subsequent amplification.

FAQ ID -1442
What QuantiFast Kit should be used on the Eppendorf Mastercycler ep realplex?

The Mastercycler ep realplex does not require ROX dye. You can use QuantiFast Probe PCR +ROX Vial Kits (no ROX dye in the master mix), QuantiFast SYBR Green PCR and QuantiFast SYBR Green RT-PCR Kits (ROX dye in the master mix does not interfere with real-time quantification).

 

FAQ ID -1436
What are the main differences between Rotor-Gene and QuantiTect or QuantiFast PCR Kits?

Rotor-Gene Kits are specifically developed for the Rotor-Gene Q PCR Cycler. The unique rotary system of the cycler combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Kits do not contain ROX dye since no normalization to a passive reference is required. Also, Rotor-Gene Kits do not contain dUTP; therefore, UNG pretreatment is not possible.

 

FAQ ID -2119
Do you offer a QuantiFast Kit for one-step RT-PCR?

Yes, we offer Quantifast one-step RT-PCR kits for Probe and SYBR Green detection:

 

FAQ ID -1695
Does the master mix in the QuantiFast Kits contain dUTP to allow UNG treatments?

No. The master mix in QuantiFast PCR Kits contains only dTTP. To perform a UNG treatment, we recommend using QuantiTect Kits.

 

FAQ ID -1431
How do QuantiFast PCR Kits compare to QuantiTect PCR Kits for quantitative real-time PCR?

We have compared QuantiFast Kits and QuantiTect Kits using around 30 different assays (using both SYBR Green and Probe detection for each assay).

QuantiFast Kits gave identical or sometimes better Ct values than QuantiTect Kits (except for very long amplicons). Therefore, scientists switching from QuantiTect to QuantiFast Kits can, in most cases, obtain comparable results.

 

 

FAQ ID -1441
How do I avoid collecting a fluorescence reading from primer-dimer with the QuantiTect SYBR Green PCR Kit?

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

 

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (Tm) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer Tm, but approximately 3ºC lower than the specific amplicon Tm.

FAQ ID -9096
Why is a 2-step (and not a 3-step) cycling protocol recommended for QuantiFast SYBR Green PCR Kits?

The two-step cycling protocols of the QuantiFast SYBR Green PCR Kits allow a significant reduction in cycling time. It is more effective than reducing individual times for annealing and extension.

 

FAQ ID -1450
Can I adjust the ROX concentration in the QuantiFast master mix?

The master mix in QuantiFast SYBR Green Kits contains an optimized concentration of ROX dye that works well with all cyclers.

QuantiFast Probe PCR Kits are available in two formats:

  • the QuantiFast Probe PCR Kit with master mix containing ROX dye
  • the QuantiFast Probe PCR +ROX Vial Kit with master mix not containing ROX dye, and a separate vial of ROX dye

We recommend using the latter with the Applied Biosystems 7500 Fast System. Use the ROX concentration indicated in the QuantiFast Probe PCR Kits handbook.

FAQ ID -1427