QuantiFast Probe PCR Kits

配列特異的プローブを用いた高速なリアルタイムPCRおよび2ステップqRT-PCR

Products

QuantiFast Probe PCR Kitsは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。
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QuantiFast Probe PCR Kit (2000)

Cat. No. / ID:  204256

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 Probe PCR Master Mix (contains ROX dye), 20 ml RNase-Free Water
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$2,436.00
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

  • 低コピーターゲットでも高感度な検出
  • 幅広いテンプレート量を正確に検出
  • 時間を最大60%まで短縮し迅速な結果
  • 至適化済みで即使用可能なマスターミックスにより、至適化せずに正確な結果を実現
  • 標準的および高速サイクラーに対応する1種類のサイクリングプロトコール

Product Details

QuantiFast Probe PCR Kitsは配列特異的なプローブを用いてリアルタイムPCRおよび2ステップRT-PCRにより迅速かつ高感度にgDNAおよびcDNAターゲットを定量します。Q-bondテクノロジーと至適化済みのマスターミックスにより、短いRamping Time機能を持つ高速サイクラーだけではなく標準的なサイクラーでも、より短時間でリアルタイムPCRを実現します。ホットスタートと即使用可能なマスターミックス中の独自のPCRバッファーシステムの組み合わせは、至適化の必要はなく、全てのリアルタイムサイクラーで高感度定量PCRを実現します。キットフォーマットは2種類あり、蛍光補正のためにROXが必要なサイクラー用QuantiFast Probe PCR Kitおよびその他のサイクラー用のQuantiFast Probe PCR +ROX Vial Kitです。マスターミックスは2~8 ℃で保存でき、簡便に取り扱えます。

Performance

QuantiFast Probe PCR Kitsでは、他のリアルタイムPCRキットよりも卓越した感度の高い結果が得られます(表"iCycler iQを用いた高感度な2ステップRT-PCR"および図" 高感度な2ステップRT-PCR")。PCR時間が最大60%短縮され(図" PCR時間を顕著に短縮")、PCRの性能を妥協しなくてもPCR結果を速く得られます(図" 感度を損なわずにより迅速な結果を実現")。従って、サンプル処理数を大幅に増やしたり、他の実験者と1台のサイクラーを効率的に共有できます。

iCycler iQを用いた高感度な2ステップRT-PCR
平均CT
ヒト白血球cDNA(ng) QuantiFast Probe PCR +ROX Vial Kit Supplier BVのキット
100 29.13 31.13
10 32.50 34.43
1 36.15 40.00
テンプレートを含まないコントロール 40.00 40.00
Primer ExpressでデザインしたIL8(ケモカイン)用TaqManアッセイを用いてtriplicateで反応を行なった。Supplier BVのキット(高速サイクリングプロトコールを使用)と比較してQuantiFast Kitでは低いCT値が得られ、わずか1 ngのcDNAからの転写物の定量が可能である。

QuantiFast Probe PCR Kitは幅広いダイナミックレンジにわたり正確な定量を実現します(図" 幅広いダイナミックレンジと高感度を実現")。テンプレートを最高107倍に希釈した溶液からでも正確な検出ができます(表"LightCycler 2.0を用いて107倍希釈したテンプレートを2ステップRT-PCRで検出"および図" 107倍希釈したテンプレートの検出")。

LightCycler 2.0を用いて107倍希釈したテンプレートを2ステップRT-PCRで検出
平均CT
Human leukocyte cDNA QuantiFast Probe PCR +ROX Vial Kit R 社Kit
100 ng 11.99 14.56
10 ng 15.54 17.99
1 ng 19.22 22.16
100 pg 22.63 25.14
10 pg 26.43 30.15
1 pg 30.02 34.43
100 fg 33.42 37.10
テンプレートを含まないコントロール 40 40.00
PCR効率 90% 80.1%
Supplier Rのβ-2ミクログロブリン用プライマーとFRETプローブのセットを用いてduplicateで反応を行なった。QuantiFast Kitは、Supplier Rのキット(高速サイクリングプロトコールを使用)と比較してCT値が低く、PCR効率が高かった。
See figures

Principle

QuantiFast Probe PCR Kitでは、標準的なサイクラー、高速サイクラーともに、広範なダイナミックレンジで非常に高感度な結果が迅速に得られます。本キットは、加水分解プローブの検出(TaqManあるいはその他のダブル標識プローブなどを含む)、FRETプローブなどの全てのタイプの配列特異的プローブを使用するためにデザインされています。新規添加剤のQ-Bondを含有する特別に開発された高速PCR用バッファーにより、変性、アニーリングおよびエクステンション時間も顕著に短縮されます(図" プライマーの高速なアニーリング")。このバッファーに入っているK+およびNH4+のイオン配合比により、プライマーとプローブが核酸テンプレートに効率的かつ安定してアニーリングし、高いPCR効率と感度を実現します(図" 特異的なプライマーアニーリング")。さらに、HotStarTaq Plus DNA Polymeraseは厳密なホットスタートを行なえるため、非特異的な産物の形成を抑えます。

2x QuantiFast Probe PCR Kitの成分*
成分 特長 利点
HotStarTaq Plus DNA Polymerase 95℃、3分間の活性化 室温での定量PCRのセットアップ
QuantiFast Probe PCR Buffer NH4+/K+イオンの配合バランス 特異性の高いプライマーのアニーリングで信頼性の高いPCR結果
ユニークなQ-Bondを含む PCR反応時間が短縮されるため迅速に結果が得られ、1日あたりの反応数を増やせる
ROX色素 Applied Biosystems社およびオプションのAgilent社の装置で蛍光シグナルを補正 ROXが必要なサイクラーで正確な定量。他のリアルタイムサイクラーでの反応を妨害しない
* dNTP Mix(dATP、dCTP、dGTPおよびdTTP)も含む。 ROX色素はマスターミックス中に含有あるいは別包装の溶液。
See figures

Procedure

QuantiFast Probe PCR Kitは反応条件やサイクリング条件の至適化が不要で即使用可能なマスターミックスです。テンプレートDNA、プライマー、プローブをマスターミックスに添加し、ハンドブックに記載されているプロトコールに従うだけで、全てのリアルタイムサイクラーで迅速で正確な結果が得られます。マスターミックス中にROXパッシブリファレンス色素が入ったキットまたは入っていないキットをお求めいただけ、事実上全てのリアルタイムサイクラーでの使用が可能です(表参照)。ROX濃度が至適化されているため、コピー数が少ない場合の検出でも自動データ解析を行なうことができます。

正しいQuantiFast Probe PCR Kitの選択ガイド
ROX色素Kit対応するサイクラー
マスターミックスに添加済みQuantiFast Probe PCR KitApplied Biosystems 7500以外のApplied Biosystemsの全てのサイクラー
別チューブで添付QuantiFast Probe PCR +ROX Vial KitApplied Biosystems 7500およびAgilent、Bio-Rad、Cepheid、QIAGEN、Eppendorf、Roche、その他のサイクラー

2ステップリアルタイムRT-PCRで最適な結果を得るためには、QuantiTect Reverse Transcription Kitを用いてcDNAを合成することをお薦めします。本キットは、ゲノムDNAコンタミの除去を組み合わせたcDNA合成をわずか20分で行ないます。 

QuantiFast Probe Assayは加水分解、プローブを用いた検出を利用したデザイン済みのゲノムワイドなアッセイです。QuantiFast Probe PCR Kitを使用して行なわれ、singleplex 2ステップqRT-PCRの結果が得られます。

Applications

QuantiFast Probe PCR Kitは、全てのリアルタイムサイクラーで、プローブを用いたcDNAターゲットの遺伝子発現解析および定量的gDNA解析に使用できます。これには、Applied BiosystemsおよびBio-Rad、Cepheid、Eppendorf、Roche、Agilent社のサイクラーを含みます。Rotor-Gene Qおよび他のRotor-Geneサイクラーを使用する場合は、これらの高速サイクリング用に特別に開発されたRotor-Gene Probe PCR Kitの使用をお勧めします。

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsProbe-based, real-time PCR, two-step RT-PCR
Sample/target typecDNA, DNA
Real-time or endpointReal-time
Reaction typeReal-time and two-step RT-PCR
Single or multiplexSingle
With or without ROXAvailable with ROX in master mix and with ROX as separate vial
SYBR Green I or sequence-specific probesSequence-specific probes
Thermal cyclerAll real-time cyclers (e.g. Roche LightCycler®, Corbett Rotor-Gene, Applied Biosystems)

Resources

MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
テクニカルインフォメーション (1)
クイックスタートプロトコール (2)
キットハンドブック (1)
For fast, quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes
サイエンティフィック・ポスター (1)
Safety Data Sheets (1)
Certificates of Analysis (1)
Kit Handbooks (1)
For fast, quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes
Quick-Start Protocols (2)

FAQ

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
Can fast cycling be carried out with QuantiFast Probe PCR Kits and FRET probes?

Yes, QuantiFast Probe PCR Kits allow the use of FRET probes, but only in 3-step cycling with a shorter initial activation step and a shorter reverse-transcription step.

A protocol is available in the QuantiFast Probe PCR Handbook.

 

 

FAQ ID -1433
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
How do Rotor-Gene Probe Kits compare with QuantiFast Probe Kits?

Rotor-Gene Probe Kits are specially developed for Rotor-Gene cyclers. The unique rotary system of the cyclers combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Probe Kits do not contain ROX dye.

 

FAQ ID -2125
Is the QuantiFast Probe PCR +ROX Vial Kit compatible with the ViiA7 cycler from Applied Biosystems?
We got comparable results on the ABI 7500 and ViiA7 using the QuantiFast Probe PCR +ROX Vial Kit master mix with low ROX concentration (ROX added to NoROX master mix) and the protocol for the ABI 7500.
FAQ ID -2651
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
How should fluorescent labeled probes be stored?

Fluorescent oligonucleotides should be stored in the dark, as light can slowly degrade the fluorescent moieties. For optimal long-term storage of fluorescent dye-labeled probes (except Cyanine 570, Cy3.5, Cyanine 670, and Cy5.5), the oligos should be resuspended in a slightly basic solution (e.g., TE buffer at pH 8.0). If resuspended below pH 7.0, the probe can degrade. We recommend to aliquot the sample, and store the aliquots at -20°C.

Note that Cyanine 570, Cy3.5, Cyanine 670, and Cy5.5 begin to degrade at a pH above pH 7.0. For best results, resuspend Cy-labeled oligos at pH 7.0, aliquot, lyophilize, and store at -20°C.

FAQ ID -784
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
Can I do singleplex PCR with the QuantiFast Probe Assays?
Yes, for 2-step RT-PCR, the QuantiFast Probe PCR Kit can be used, for 1-step RT-PCR, the QuantiFast Probe RT-PCR Plus Kit is combined with the assays.
FAQ ID -2362
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
Are TaqMan® Gene Expression Assays from Applied Biosystems compatible with QuantiFast Probe PCR Kits?

Yes, use the ABI gene expression assays at a final concentration of 1x with the QuantiFast Probe PCR Kits.

See trademarks

FAQ ID -1452
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
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
What is the detection limit of the QuantiFast Kits for real-time PCR?

The QuantiFast SYBR Green PCR and QuantiFast Probe PCR Kits allow reliable detection down to 10 target copies. Detection of lower copy numbers down to single copy level may also be possible, however, it depends on the stochastics when working with highly diluted samples. Additional optimization of primer/probe design is usually required.

FAQ ID -1432
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
Which real-time PCR kits are recommended downstream of the QuantiTect Whole Transcriptome Kit?

We highly recommend any QuantiTect or QuantiFast Kit for quantitative PCR on cDNA generated with the QuantiTect Whole Transcriptome Kit.

 

FAQ ID -1592
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
Can the QuantiFast Probe PCR +ROX Vial Kit be used on the ABI PRISM 7900 and Applied Biosystems 7500 cyclers?

The ABI PRISM 7900 requires a high ROX concentration, which cannot be achieved with the 50x ROX dye solution supplied in the QuantiFast Probe PCR +ROX Vial Kit. However, this kit provides the optimal ROX concentration for the Applied Biosystems 7500.

 

FAQ ID -1555
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
Are there problems with the default threshold setting on the Applied Biosystems 7500 when using QuantiFast Probe PCR Kits?

No. We recommend using QuantiFast Probe +ROX Vial Kits: the master mix does not contain ROX dye, but the kit is supplied with a separate tube of ROX dye. The kits allow to adjust the ROX concentration in the master mix according to your cycler’s requirements.

Please follow specific recommendations in the QuantiFast Probe PCR Handbook.

 

FAQ ID -1702
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
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
Is it necessary to perform calibration steps for the use of the MAX dye in duplex RT-PCR experiments on different cyclers?

It is not necessary to perform calibration steps with MAX dye.  For instruments from Applied Biosystems, simply use the VIC channel/filter for the detection of MAX. The emission maxima of MAX and VIC are very similar (557nm and 554nm), respectively. On the Rotor-Gene Q, use the yellow channel. On the BioRad CFX, use channel 2 for MAX detection. On the LightCycler 480, use the combination 523 nm (Excitation) / 568 nm (Emission).  MAX was chosen as a second dye label because VIC label is protected by ABI and HEX showed more crosstalk in our experiments.

FAQ ID -2373
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
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
Can the QuantiFast Probe PCR +ROX Vial mastermix be stored long-term after adding ROX dye?

Yes, when working with the QuantiFast Probe PCR +ROX Vial Kit, please add the volume of ROX dye solution specified in the QuantiFast Probe PCR Handbook.

Master mix containing ROX dye can be stored for up to 2 months at 4°C.

 

FAQ ID -1435