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10.1 End-point PCR and RT-PCR 10.2 Real-time PCR and RT-PCR 10.3 Cloning
www.qiagen.com
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PCR assay technologies provide highly sensitive detection of DNA or RNA. Robust PCR systems allow amplification and detection without the need for user optimization of reaction conditions. PCR-based molecular testing of animals is increasingly important for many veterinary and agricultural applications. QIAGEN provides the solutions you need from sample to result. www.qiagen.com/PG/PCR
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www.qiagen.com/PG/PCR
PCR enzymes
Standard PCR, fridge storage Standard PCR Standard PCR, with dNTPs Hot-start PCR TopTaq DNA Polymerase Taq DNA Polymerase Taq PCR Core Kit HotStarTaq Plus DNA Polymerase HotStarTaq DNA Polymerase 455 457 457 460 462
dNTPs
PCR grade dNTP Set, PCR Grade dNTP Mix, PCR Grade 467 467
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n RT-PCR kits
n Sensitive one-step RT-PCR in one tube n Accurate and long-range two-step RT-PCR QIAGEN OneStep RT-PCR Kit QIAGEN LongRange 2Step RT-PCR Kit 474 475
n Reverse-transcription enzymes
n Using 50 ng to 2 g RNA n Using less than 50 ng RNA Omniscript RT Kit Sensiscript RT Kit 477 478
n Automated solutions
n Reaction setup in all formats n Multicapillary electrophoresis of PCR products n HRM analysis
New
QIAgility
67 83 78 362 256
QIAxcel System
New
Rotor-Gene Q
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n Real-time PCR and RT-PCR: ultrafast, precise quantification on Rotor-Gene cyclers n Real-time PCR and RT-PCR: fast cycling n Real-time PCR and RT-PCR: standard cycling n Real-time RT-PCR: direct from cell lysates without RNA purification
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QuantiFast SYBR Green Kits QuantiTect SYBR Green Kits miScript PCR System
QuantiFast Probe Kits QuantiTect Probe Kits QuantiTect Virus Kits Type-it Fast SNP Probe PCR Kit EpiTect MethyLight PCR Kits EpiTect MethyLight Assays
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n Standard cycling with optional UNG pretreatment n Viral DNA and RNA detection
513 505
516 165
n Automated solutions
n RNA purification and RT-PCR setup in 96-well format n Reaction setup in all formats n Real-time PCR cycler and HRM analyzer BioRobot Universal System
New New
62 67 78
QIAgility Rotor-Gene Q
10.3 Cloning
n Cleanup of PCR products n Cloning of PCR products
n With competent cells n Without competent cells See PCR cleanup
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518 519
The PCR assay technologies presented in this chapter (with the exceptions of TopTaq DNA Polymerase, the TopTaq Master Mix Kit, the PyroMark PCR Kit, and the Type-it HRM PCR Kit) are intended for research use. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The PyroMark PCR Kit, TopTaq DNA Polymerase, the TopTaq Master Mix Kit, and Type-it HRM PCR Kit are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
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Q
Applications Standard PCR Standard PCR, hot-start High-fidelity PCR Fast-cycling PCR, hot-start Long-range PCR Multiplex PCR Genotyping (STR, microsatellites) Amplification of SNP loci Genotyping (mutation detection) Single-cell PCR Methylation-specific PCR (MSP) Nested PCR DNA virus detection Page : Recommended product.
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EN
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Q
Benefits Speed Advantages Ultrafast PCR Multiplex PCR Hot-start (5 min) PCR performance Hot-start (15 min) Hot-start (5 min) Maximal specificity Maximal sensitivity Q-Solution Ease of use and convenience Direct UA/ TA cloning PCR with gel tracking dyes Roomtemperature setup Complete kit with dNTPs Master mix with dNTPs Fridge storage Page n
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EN
n n
456 462
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460
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n: Recommended product. (All products shown provide reliable results without the need for optimization of PCR parameters.) * Amplification product size: 3.5 kb.
Amplification product size: 5 kb. Amplification product size: 1.5 kb. Amplification product size: 40 kb.
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EN IA Q G
Applications Two-step RT-PCR* One-step RT-PCR* Long-range, two-step RT-PCR Single-cell, one-step RT-PCR Virus detection Page : Recommended product.
IA
RT-PCR
Direct UA/TA cloning Complete kit format including nucleotides Fast and easy procedure
475
(All products shown provide reliable results without the need for optimization of PCR parameters.) * Amplification product size: 12.5 kb.
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IA
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10.1
A
QIAGEN M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M
4C storage for immediate reaction setup Convenient room-temperature handling A single preoptimized protocol suitable for all PCR assays High yields of amplified DNA
Product description TopTaq DNA Polymerase combines a number of convenient and unique features to provide unrivaled ease-of-use and ensure high DNA yields in a wide range of PCR applications. The unique TopTaq Stabilizer included in the enzyme storage buffer allows storage of TopTaq DNA Polymerase at 4C and reaction setup at room temperature without time-consuming thawing of reagents. CoralLoad Concentrate, containing 2 gel-tracking dyes, is also provided, enabling immediate loading of PCR products. High yields of PCR products are achieved, even when amplifying a range of different sized products using the same Mg2+ concentrations and annealing temperatures (see figure). Applications TopTaq DNA Polymerase is highly suited to all end-point PCR applications. TopTaq DNA Polymerase is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
B
QIAGEN Supplier A Supplier I Supplier T 11 12 13 14 11 12 13 14 11 12 13 14 11 12 13 14
High PCR product yields without optimization. A TopTaq DNA Polymerase was used to amplify 14 different PCR products from mammalian genomic DNA ranging in size from 100 bp to 2 kb according to the standard preoptimized protocol and using identical cycling conditions. B The same 4 PCR products amplied in lanes 1114 in Figure A were amplied under identical cycling conditions using DNA polymerases from the indicated suppliers. TopTaq DNA Polymerase (QIAGEN) showed high yields of specic PCR products regardless of fragment size.
Related products
Automated PCR setup QIAgility Automated DNA fragment analysis QIAxcel System 67 83
TopTaq DNA Polymerase (1000 U)* TopTaq DNA Polymerase (5000 U)*
* TopTaq DNA Polymerase is a proprietary recombinant polymerase originally isolated from a thermophilic eubacterium. For dNTPs, see page 467. Includes 250, 1000, or 5000 units TopTaq DNA Polymerase, 10x PCR Buffer (containing 15 mM MgCl2), 10x CoralLoad Concentrate, 5x Q-Solution, and 25 mM MgCl2. Further information and online ordering: www.qiagen.com/PG/TopTaqDNA
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A
M 20C 4C 25C M
Ready-to-use master mix with minimal pipetting steps Fridge storage eliminating freezethaw cycles Reliable high-yield PCR performance Convenient room-temperature handling Optimized protocol suitable for all PCR assays
Product description
B
20C
A01 A02 A03 A04 A05 B00
4C
A06 A07 A08 A09
25C
A10 A11 A12
10000.0 5000.0 2000.0 1000.0 700.0 500.0 400.0 300.0 200.0 100.0 15.0
The TopTaq Master Mix Kit offers maximum convenience by combining all the benefits of TopTaq DNA Polymerase with the advantage of a ready-to-use master mix. It contains TopTaq DNA Polymerase, the unique QIAGEN PCR Buffer that minimizes the requirement for optimization, and dNTPs. The unique TopTaq Stabilizer included in the enzyme storage buffer allows storage of TopTaq Master Mix at 4C and reaction setup at room temperature without time-consuming thawing of reagents. Providing all components in a ready-to-use master mix reduces pipetting steps, which lowers the risk of contamination. High yields of PCR product are achieved, even after storing the TopTaq Master Mix for 4 months at 25C, 4C, or 20C. Applications The TopTaq Master Mix Kit is highly suited to all end-point PCR applications. The TopTaq Master Mix Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Reliable high-yield PCR independent of storage conditions. PCR was performed using TopTaq Master Mix stored at 20C, 4C, and 25C for 4 months. Two human genes (a 750 bp fragment of the prp gene, and a 1200 bp fragment of the hugl gene) were amplied according to the standard optimized protocol (duplicates shown). The results show high yields of specic PCR product. A Agarose gel analysis (M: GelPilot 1 kb Plus Marker). B Gel image produced by the QIAxcel System. Markers: GelPilot 1 kb Plus Marker and QX Alignment Marker 15 bp/10 kb.
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Automated PCR setup Automated DNA fragment analysis QIAgility QIAxcel System 67 83
* Includes 2x TopTaq Master Mix (with 250 units of TopTaq DNA Polymerase, 3 mM MgCl2, and 400 M each dNTP), 10x CoralLoad Concentrate, and RNase-free water. Further information and online ordering: www.qiagen.com/PG/TopTaqMasterMix
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M A07 A07
A A08 A09
B A10 A11
C A12
QIAGEN PCR Buffer for minimal optimization Additional ready-to-load PCR buffer for faster handling Q-Solution for amplification of GC-rich templates Choice of formats for convenience and ease of handling
Product description Taq DNA Polymerase is supplied with the unique QIAGEN PCR Buffer that minimizes the requirement for optimization and Q-Solution, a novel additive that enables efficient amplification of difficult (e.g., GC-rich) templates (for more information, see page 463). In addition, CoralLoad PCR Buffer (containing 2 gel-tracking dyes) is also provided, enabling immediate loading of PCR products. The Taq PCR Core Kit also includes a dNTP mix. Applications Taq DNA Polymerase is suitable for standard and specialized applications, including:
High-resolution analysis of amplicons using the QIAxcel. PCR products were generated using QIAGEN Taq DNA Polymerase and analyzed on the QIAxcel with the QIAxcel DNA Screening Gel Cartridge. Duplicates of 3 different amplicons are shown. A: 100 bp. B: 500 bp. C: 1000 bp. M: phiX/HaeIII Marker.
General PCR RT-PCR Differential display PCR-based DNA fingerprinting (VNTR, STR, and RAPD)
Related products
Automated PCR setup Automated DNA fragment analysis Molecular weight markers QIAgility QIAxcel System GelPilot Molecular Weight Markers 67 83 582 Ready-to-load PCR buffer. A The novel CoralLoad PCR Buffer B containing 2 gel-tracking dyes allows the PCR product to be loaded immediately onto an analytical gel for increased speed and convenience.
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Product Taq DNA Polymerase (250 U)* Taq DNA Polymerase (1000 U)* Taq DNA Polymerase (5000 U)* Taq DNA Polymerase (25,000 U)* Taq PCR Core Kit (250 U) Taq PCR Core Kit (1000 U)
* For dNTPs, see page 467.
For 800 x 50 l reactions For 4000 x 50 l reactions For 20,000 x 50 l reactions For 200 x 50 l reactions
Includes 250, 1000, 5000, or 25,000 units of Taq DNA Polymerase, 10x PCR Buffer (contains 15 mM MgCl2), 10x CoralLoad PCR Buffer (contains 15 mM MgCl2), 5x Q-Solution, and 25 mM MgCl2. Also includes dNTP Mix (containing 10 mM each dNTP).
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M C
10
20
50
copies
Reproducible PCR. A fragment of the hepatitis B surface antigen gene (gene S) was amplied from 10, 20, and 50 copies of target template, using the Taq PCR Master Mix Kit. Five parallel amplications were performed for each amount of starting template DNA. Equal volumes of the PCR products were analyzed on a 2% agarose gel. C: negative control; M: markers.
General PCR RT-PCR Differential display PCR-based DNA fingerprinting (VNTR, STR, and RAPD)
Related products
Automated PCR setup Automated DNA fragment analysis Molecular weight markers QIAgility QIAxcel System GelPilot Molecular Weight Markers 67 83 582
Product Taq PCR Master Mix Kit (250 U) Taq PCR Master Mix Kit (1000 U)
* Includes Taq PCR Master Mix (with 1.5 mM MgCl2 and 200 M each dNTP), containing 250 or 1000 units of Taq DNA Polymerase, and RNase-free water. Further information and online ordering: www.qiagen.com/PG/TaqPCRMasterMix
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Pl
us q Ta
Fast 5-minute enzyme activation time High PCR specificity with minimal optimization Ready-to-load PCR buffer for faster and easier handling
Product description The polymerase combines the high specificity, sensitivity, and minimal optimization of HotStarTaq DNA Polymerase (see page 462) with a fast 5-minute activation time. The novel CoralLoad PCR buffer containing gel-tracking dyes and room-temperature setup further streamlines the PCR procedure. Standard PCR buffer is also included for your convenience. In addition, Q-Solution, a novel additive that enables efficient amplification of difficult (e.g., GC-rich) templates (for more information, see page 463), is also provided. Applications HotStarTaq Plus DNA Polymerase is suitable for general PCR, complex genomic or cDNA templates, and very low-copy targets (e.g., single-cell PCR).
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Automated PCR setup Automated DNA fragment analysis Molecular weight markers QIAgility QIAxcel System GelPilot Molecular Weight Markers 67 83 582
Highest specificity with HotStarTaq Plus Polymerase. PCR was carried out using QIAGEN HotStarTaq Plus, HotStarTaq, and Taq DNA Polymerases and 3 hot-start PCR enzymes from the indicated suppliers. Parallel reactions were performed following the suppliers recommendations, using 50 ng human genomic DNA. A 1.5 kb fragment of the human CFTR gene was amplied in 35 PCR cycles. M: markers.
HotStarTaq Plus DNA Polymerase (1000 U)* HotStarTaq Plus DNA Polymerase (5000 U)*
* For dNTPs, see page 467. Polymerase supplied in single tube. Includes 250, 1000, 5000, or 25,000 units of HotStarTaq Plus DNA Polymerase, 10x PCR Buffer (contains 15 mM MgCl2), 10x CoralLoad PCR Buffer (contains 15 mM MgCl2), 5x Q-Solution, and 25 mM MgCl2. Further information and online ordering: www.qiagen.com/PG/HotStarTaqPlusDNA
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Fast 5-minute enzyme activation time Fewer pipetting steps reduce the risk of contamination Higher PCR specificity and reduced nonspecific amplification Optional ready-to-load buffer additive for easier handling
Product description HotStarTaq Plus Master Mix contains HotStarTaq Plus DNA Polymerase, the unique QIAGEN PCR Buffer that minimizes the requirement for optimization (for more information, see page 463), and dNTPs. The HotStarTaq Plus Master Mix Kit provides the same unrivaled highly specific and sensitive PCR as the HotStarTaq Master Mix Kit combined with a fast 5-minute enzyme activation time. In addition, CoralLoad Concentrate, containing 2 gel-tracking dyes, is also provided and can be added to the master mix to enable immediate loading of PCR products. Applications The HotStarTaq Plus Master Mix Kit is suitable for general PCR, complex genomic templates, complex cDNA templates, and very low-copy targets (e.g., single-cell PCR).
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Automated PCR setup Automated DNA fragment analysis Molecular weight markers QIAgility QIAxcel System GelPilot Molecular Weight Markers 67 83 582 HotStarTaq Plus Master Mix Kit (2500). Bulk-size kit containing single tube of 2.5 ml master mix.
Ready-to-load PCR buffer. A The novel CoralLoad Concentrate B containing 2 gel-tracking dyes allows the PCR product to be loaded immediately onto an analytical gel for increased speed and convenience.
Product HotStarTaq Plus Master Mix Kit (250)* HotStarTaq Plus Master Mix Kit (1000) HotStarTaq Plus Master Mix Kit (2500)*
* Master mix supplied in single tube.
Contents For 250 x 20 l reactions For 1000 x 20 l reactions For 2500 x 20 l reactions
Includes HotStarTaq Plus Master Mix (with 1.5 mM MgCl2 and 200 M each dNTP), containing 250, 1000, or 2500 units HotStarTaq Plus DNA Polymerase, 10x CoralLoad Concentrate, and RNase-free water.
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Distribute
Highly specific PCR Reduced nonspecific amplification Easy handling and room-temperature setup
Amplification
TC H UG L N TC p5 3
TC H UG
p5
C04 C05 C06 C07 C08 C09 C04 C05 C06 C07 C08 C09
TC
Product description HotStarTaq DNA Polymerase uses a chemically mediated hot-start that, unlike antibody-mediated systems, leads to complete inactivation of the polymerase until the initial heat activation step. HotStarTaq DNA Polymerase is supplied with unique QIAGEN PCR Buffer, which minimizes nonspecific amplification products, primerdimers, and background. Q-Solution, a novel additive that enables efficient amplification of GC-rich templates and templates with a high degree of secondary structure, is also provided. Principle HotStarTaq DNA Polymerase is a modified form of the recombinant 94 kDa Taq DNA Polymerase from QIAGEN. HotStarTaq DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primerdimers at low temperatures. HotStarTaq DNA Polymerase is activated by a 15-minute, 95C incubation step, which can easily be incorporated into existing thermal cycling programs.
100.0
15.0
Manual
QIAgility
Highly specific PCR results with both manual and automated PCR setup. PCR setup was performed manually or was automated using the QIAgility. PCR products were analyzed on the QIAxcel. HotStarTaq DNA Polymerase resulted in specic amplication of both p53 (400 bp) and HUGL (1200 bp). NTC: no template control.
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HotStarTaq DNA Polymerase provides high PCR specificity and often increases the yield of the specific PCR product. The balanced combination of K+ and NH4+ used in the QIAGEN PCR Buffer strongly increases primer annealing specificity. The improved specificity is caused by ammonium ions destabilizing the weak hydrogen bonds at mismatched bases. The use of this buffer also often eliminates the need for optimization of Mg2+ concentration or annealing temperature for different primertemplate systems and maintains high primer annealing specificity in each PCR cycle. This novel buffer is provided with all QIAGEN PCR kits (pages 455475) for increased specificity. HotStarTaq DNA Polymerase is provided with Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. This unique reagent will often enable or improve a suboptimal PCR caused by difficult templates that, for example, have a high degree of secondary structure or templates that are GC-rich. Unlike other commonly used PCR additives, Q-Solution is used at just one working concentration, it is nontoxic, and PCR purity is guaranteed. Procedure Due to the hot start, PCR setup is quick and convenient as all reaction components can be combined at room temperature. The HotStarTaq procedure makes hot-start PCR simple and easy, eliminating the extra handling steps and contamination risks associated with conventional hot-start methods. Applications HotStarTaq DNA Polymerase is highly suitable for amplification reactions involving complex genomic or cDNA templates, multiple primer pairs, or very low-copy targets (e.g., single-cell PCR).
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Automated PCR setup Automated DNA fragment analysis dNTPs Molecular weight markers QIAgility QIAxcel System
Stabilization
K+
Destabilization NH 4+
NH3 + H+
Stabilization
K+
P K+
P K+
Weak hydrogen band (e.g., between C and T nucleotides) Strong hydrogen band (e.g., between A and T nucleotides)
NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing principally the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specic to nonspecic primertemplate binding over a wide temperature range.
67 83 467 582
dNTP Set, PCR Grade and dNTP Mix, PCR Grade GelPilot Molecular Weight Markers
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Amplification of difficult genomic loci Amplification of difficult SNP loci High-throughput/fast PCR Increased sensitivity for single-cell PCR
Highly specific hot-start PCR and HRM analysis Probe-based real-time PCR Fast-cycling PCR Highly specific hot-start PCR
Type-it Mutation Detect PCR Kit Type-it Microsatellite PCR Kit QIAGEN Multiplex PCR Kit Type-it HRM PCR Kit Type-it Fast SNP Probe PCR Kit QIAGEN Fast Cycling PCR Kit HotStarTaq Master Mix Kit HotStarTaq Plus DNA Polymerase HotStarTaq Plus Master Mix Kit HotStar HiFidelity Polymerase Kit QIAGEN LongRange PCR Kit EpiTect MSP Kit EpiTect HRM PCR Kit QIAGEN OneStep RT-PCR Kit QIAGEN LongRange 2Step RT-PCR Kit TopTaq DNA Polymerase TopTaq Master Mix Kit Taq DNA Polymerase Taq PCR Core Kit Taq PCR Master Mix Kit
Sequence accuracy Amplification of long PCR products Detection of methylated DNA Increased sensitivity
High-fidelity PCR Long-range PCR Methylation-specific PCR Methylation analysis using HRM analysis One-step RT-PCR Long-range two-step RT-PCR
Standard PCR
End-point PCR
Product HotStarTaq DNA Polymerase (250 U)* HotStarTaq DNA Polymerase (1000 U) HotStarTaq DNA Polymerase (5000 U)* HotStarTaq DNA Polymerase (25,000 U)
* Polymerase supplied in single tube.
203209
Includes 250, 1000, 5000, or 25,000 units of HotStarTaq DNA Polymerase, 10x PCR Buffer (contains 15 mM MgCl2), 5x Q-Solution, and 25 mM MgCl2.
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Easy reaction setup at room temperature Ready to use with fewer pipetting steps High PCR specificity and reduced nonspecific amplification
Product description HotStarTaq Master Mix contains HotStarTaq DNA Polymerase, the unique QIAGEN PCR Buffer that minimizes the requirement for optimization (for more information, see page 463), and dNTPs. Providing all components in a master mix reduces pipetting steps and risk of contamination, while increasing throughput and reproducibility. Applications The HotStarTaq Master Mix Kit is highly suitable for amplification reactions involving complex genomic or cDNA templates, multiple primer pairs, or very low-copy targets (e.g., single-cell PCR).
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Automated PCR setup Automated DNA fragment analysis Molecular weight markers QIAgility QIAxcel System GelPilot Molecular Weight Markers 67 83 582 HotStarTaq Master Mix Kit.
Product HotStarTaq Master Mix Kit (250 U)* HotStarTaq Master Mix Kit (1000 U) HotStarTaq Master Mix Kit (2500 U)*
* Master mix supplied in single tube.
Contents For 100 x 50 l reactions For 400 x 50 l reactions For 1000 x 50 l reactions
Includes HotStarTaq Master Mix (with 1.5 mM MgCl2 and 200 M each dNTP), containing 250, 1000, or 2500 units of HotStarTaq DNA Polymerase, and RNase-free water.
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35 cycles
35 cycles
Hot-start PCR amplification in as little as 15 minutes (35 cycles) Ideally suited for use with any thermal cycler Ready-to-use master mix with HotStarTaq Plus Polymerase No need to redesign primers for fast PCR Optional ready-to-load PCR dye for easier handling
Product description
Final extension Total time around 20 minutes
The fast-cycling PCR master mix contains HotStarTaq Plus DNA Polymerase for highly specific and sensitive PCR, and a unique buffer formulation for extremely short denaturation, annealing, and extension steps. The new patent-pending PCR buffer significantly reduces the time required to form the polymerase, primer, and template complex, reducing the total PCR cycling time from approximately 1.5 hours to just 20 minutes. The optional CoralLoad Dye contains gel-tracking dyes for convenient analysis. In addition, Q-Solution, a novel additive that enables efficient amplification of difficult (e.g., GC-rich) templates (for more information, see page 463), is also provided. The QIAGEN Fast Cycling PCR Kit in combination with the QIAxcel results in time savings of over 75%. Applications
Final extension Total time >1 hour
The kit is suitable for general PCR, complex genomic templates, and complex cDNA templates. The kit can be used with standard as well as fast-ramping thermal cyclers.
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Automated PCR setup Automated DNA fragment analysis QIAgility QIAxcel System 67 83
Product QIAGEN Fast Cycling PCR Kit (200)* QIAGEN Fast Cycling PCR Kit (1000)
* Master mix supplied in single tube. Includes 2x QIAGEN Fast Cycling PCR Master Mix (with optimized MgCl2 concentration and 200 M each dNTP), containing HotStarTaq Plus DNA Polymerase, 10x CoralLoad Dye, 5x Q-Solution, and RNase-free water. Further information and online ordering: www.qiagen.com/PG/FastCyclingPCR
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Individual dNTPs or premixed ready-to-use solution Highly pure Suitable for standard and highly sensitive PCR applications
Product description dNTP Set, PCR Grade is a complete set of individual, highly pure dNTPs. Each individual 100 mM dNTP is supplied in water and can be diluted and mixed with any other dNTP to the desired concentration. dNTP Mix, PCR Grade contains premixed dATP, dCTP, dGTP, and dTTP in water (pH 7.5), each at a concentration of 10 mM. Highly pure dNTPs are important for successful PCR, as the presence of contaminating impurities in PCR can result in a decrease in amplification sensitivity and product yield. Applications QIAGEN dNTPs are suitable for use in all standard PCR techniques and all sensitive PCR techniques such as long-range PCR, multiplex PCR, and RT-PCR. In addition, these products are ideal for use in combination with all common PCR and RT-PCR enzymes, including QIAGEN Taq DNA Polymerase, HotStarTaq Plus DNA Polymerase, and reverse-transcription enzymes.
dNTP Set, PCR Grade.
Product dNTP Mix, PCR Grade (200 l) dNTP Mix, PCR Grade (800 l) dNTP Set, PCR Grade, 4 x 100 l dNTP Set, PCR Grade, 4 x 250 l
* Mix containing 10 mM each of dATP, dCTP, dGTP, and dTTP.
Contents For 200 x 50 l reactions* For 800 x 50 l reactions* For 1000 x 50 l reactions
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M 10 1 10 1 10 1 10 1 10 1 ng
High sensitivity and specificity Unique UA/TA cloning feature 10-fold higher fidelity than Taq DNA Polymerase Room-temperature setup and fast enzyme activation
Product description The ready-to-use, optimized kit includes enzyme, buffers, and dNTPs. HotStar HiFidelity DNA Polymerase provided in the kit is a hot-start proofreading enzyme uniquely modified to produce A overhangs, enabling direct and streamlined UA/TA cloning. The buffer contains Factor SB to prevent degradation of primers and template during PCR setup, providing highly sensitive and reliable high-fidelity PCR. In addition, Q-Solution enables efficient amplification of difficult (e.g., GC-rich) templates (for more information, see page 463). Applications HotStar HiFidelity DNA Polymerase provides 10-fold higher fidelity than Taq DNA Polymerase combined with a unique UA/TA cloning feature, enabling use in highly sensitive applications, including RT-PCR of full-length transcripts, direct cloning, and mutation analysis.
Highly sensitive and reliable PCR. PCR was carried out using HotStar HiFidelity DNA Polymerase (QIAGEN) and 4 high-delity PCR enzymes from the indicated suppliers. A 2.3 kb fragment of the human IL9R gene was amplied from genomic DNA in 40 PCR cycles. M: markers. * Two different high-delity enzymes from Supplier I.
Efficient TA/UA cloning. A 955 bp PCR fragment was amplied from 100 ng human genomic DNA using the HotStar HiFidelity Polymerase Kit and standard Taq DNA Polymerase. 4 l of each PCR product was cloned using either a commercially available TA or UA cloning kit. Blue/white screening was used to determine the cloning efciency.
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Automated PCR setup Automated DNA fragment analysis Molecular weight markers QIAgility QIAxcel System GelPilot Molecular Weight Markers 67 83 582
Product HotStar HiFidelity Polymerase Kit (100 U) HotStar HiFidelity Polymerase Kit (1000 U)
* Includes 100 or 1000 units of HotStar HiFidelity DNA Polymerase, 5x PCR Buffer (including dNTPs, Factor SB, and an optimized concentration of MgSO4), 5x Q-Solution, 25 mM MgSO4, and RNase-free water. Further information and online ordering: www.qiagen.com/PG/HotStarHiFidelity
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Amplification of extremely long PCR products (up to 40 kb) Low error rates, ensured by high-fidelity enzyme Minimal PCR optimization due to unique buffer system Amplification of low-copy targets and GC-rich templates
Product description The ready-to-use QIAGEN LongRange PCR Kit contains a blend of Taq DNA polymerase and a powerful high-fidelity enzyme to ensure high amplification efficiency and maximum fidelity. The unique PCR buffer enhances extension rates and fidelity, even when amplifying complex templates. PCR products of up to 40 kb can be reliably amplified. Q-Solution, for amplification of GC-rich templates (for more information, see page 463), and high-purity dNTPs are also provided. Applications The QIAGEN LongRange PCR Kit is suitable for high-fidelity long-range PCR of standard and complex templates for all applications, including cloning and sequencing. For long-range two-step RT-PCR, see page 475.
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Automated PCR setup Molecular weight markers QIAgility GelPilot Molecular Weight Markers 67 582
Q IA G E Su N pp li e rR Su pp li e rT
High yields of very long PCR products. 27 kb and 24 kb PCR products were amplied from complex DNA using the QIAGEN LongRange PCR Kit and 2 long-range PCR kits from the indicated suppliers according to the manufacturers instructions. The QIAGEN LongRange PCR Kit provided much higher yields of specic product than the other kits tested.
Product QIAGEN LongRange PCR Kit (20) QIAGEN LongRange PCR Kit (100) QIAGEN LongRange PCR Kit (250)
* Includes LongRange PCR Enzyme Mix (40, 200, or 500 units), LongRange PCR Buffer, 5x Q-Solution, RNase-free water, and 10 mM dNTPs. Further information and online ordering: www.qiagen.com/PG/LongRangePCR Product Guide 201011 Sample & Assay Technologies 469
Q IA G E Su N pp li e rR Su pp li e rT
27 kb 24 kb
10.1
955 bp
No optimization required High specificity and sensitivity with a built-in hot start Highly suited for many types of multiplex PCR applications Easy to use and cost-effective
Product description
99 bp
Successful 19-plex PCR without optimization. Multiplex PCR of 19 targets (99955 bp) was carried out for 35 cycles using standard conditions (Std) for the QIAGEN Multiplex PCR Kit (QIAGEN) without optimization, or using the indicated Mg2+ concentrations with a hot-start enzyme and supplied KCl-based buffer from Supplier I (Supplier I). The QIAGEN Multiplex PCR Kit allowed amplication of all 19 fragments in parallel without the need for optimization. M: markers.
The QIAGEN Multiplex PCR Kit provides QIAGEN Multiplex PCR Master Mix with HotStarTaq DNA Polymerase and a unique PCR buffer containing the novel synthetic Factor MP. Together with optimized salt concentrations, Factor MP stabilizes specifically bound primers and enables efficient extension of all primers in the reaction without optimization (see figure). Q-Solution, a novel additive that enables efficient amplification of difficult (e.g., GC-rich) templates (for more information, see page 463), is also supplied. Applications The QIAGEN Multiplex PCR Kit is highly suitable for use in many types of multiplex PCR applications such as microsatellite analysis, genotyping, GMO typing, and SNP amplification.
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Product QIAGEN Multiplex PCR Kit (100) QIAGEN Multiplex PCR Kit (1000)
* Includes QIAGEN Multiplex PCR Master Mix (containing HotStarTaq DNA Polymerase, Multiplex PCR Buffer, MgCl2 [nal concentration 3 mM], and dNTPs), 5x Q-Solution, and RNase-free water.
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New
Specific amplification of bisulfite-converted DNA Specific amplification of genomic DNA from various sources Optimized for successful Pyrosequencing analysis Consistently high yields of PCR product Convenient master mix format and optimized protocols
Product description The PyroMark PCR Kit is specifically optimized for Pyrosequencing analysis. It ensures highly specific amplification of template DNA from various sources for a range of Pyrosequencing applications. These include mutation detection, SNP analysis, methylation analysis, and sequencing. The kit is provided in a convenient master mix format consisting of HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer. In addition, Q-Solution, a novel additive that enables efficient amplification of difficult or GC-rich templates (see page 463), is also provided. The kit is supplied with CoralLoad Concentrate for added convenience and improved Pyrosequencing performance. The concentrate contains 2 gel-tracking dyes for immediate loading of PCR products for agarose gel analysis. Applications The PyroMark PCR Kit is used for template amplification for subsequent Pyrosequencing analysis. The PyroMark PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
E S C T A C T A CGA T A T A T C A G A T CGA C T GA 5 10 15 20 25
Standard PCR
B8: TACTCRTAAATACRACRCTTATATTCTTAAAAAAAATTTTAC
130 120 110 100 90 80 70 60 50 40 30 20 10 0
54%
57%
48%
E S C T AC T A C G A T A T AT C AG A T C G A C T GA 15 20 10 5 25
PCR amplification optimized for Pyrosequencing analysis. The PyroMark PCR Kit ensures greater amplication efciency with high specicity. This leads to higher peaks and more reliable Pyrosequencing results compared to standard PCR.
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Bisulfite conversion EpiTect Bisulfite Kits of DNA Pyrosequencing analysis P yroMark Systems 159
72
* Includes PyroMark PCR Master Mix (contains HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing dNTPs and 3 mM MgCl2 ), 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-free water. Further information and online ordering: www.qiagen.com/PG/PyroMarkPCR Product Guide 201011 Sample & Assay Technologies 471
10.1
FAM Channel
5000 4000 3000 2000 1000 0 80
130 160 200 240 280 320
Reliable microsatellite analysis by multiplex PCR Microsatellite assay development without optimization Successful and specific coamplification of all fragments Optimized protocol for fast and reliable results
Product description The Type-it Microsatellite PCR Kit is based on highly specific HotStarTaq Plus DNA Polymerase and a patented buffer system, both of which enable reliable multiplex PCR-based microsatellite analysis without optimization. The combination of all components provided in the master mix and the specialized formulation result in highly specific amplification of all loci in parallel. Optimized protocols are also provided to enable subsequent analysis either by high-resolution capillary sequencing or by using other electrophoresis instruments.
NED Channel
5000 4000 3000 2000 1000 0
80
130
160
200
240
280
320
Reliable 13-plex STR analysis using the Type-it Microsatellite PCR Kit. Only the FAM and NED channel representing 7 of 13 analyzed human STR loci of the respective 4 channels of a 3730 xl Capillary Sequencer (Applied Biosystems) are shown.
Applications The Type-it Microsatellite PCR Kit is dedicated for fast and reliable microsatellite analysis using fluorescent or nonfluorescent primers. Reliable typing of humans, animals, plants, and bacteria using microsatellites, STR, or VNTR markers is achieved without the need for lengthy optimization procedures.
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Automated PCR setup QIAgility 67
Product Type-it Microsatellite PCR Kit (70) Type-it Microsatellite PCR Kit (200)
* Includes Type-it Multiplex PCR Master Mix (with optimized MgCl2 concentration and dNTPs), containing HotStarTaq Plus DNA Polymerase, 5x Q-Solution, and RNase-free water. Further information and online ordering: www.qiagen.com/PG/TypeitMicrosatellite
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10.1
Types of mutations Deletions Translocation Duplications Insertions SNPs Unclassified Fields of research Typing of disease Ioci GMO analysis Typing of transgenic plants/animals Bacterial typing SNP preamplification (SNaPshot Multiplex Kit)
Successful and reproducible analysis of multiple mutations Multiplex PCR assay development without optimization Specific and sensitive coamplification of all fragments Optimized protocol for fast and reliable results
Product description The Type-it Mutation Detect PCR Kit is based on highly specific HotStarTaq Plus DNA Polymerase and a patented buffer system, both of which enable reliable amplification of mutant loci by multiplex PCR without optimization. The combination of all components provided in the master mix and the dedicated protocol result in highly specific amplification of all fragments in parallel. Subsequent analysis is straightforward and easy and can be carried out on agarose gels, automated electrophoresis instruments, and also by high-resolution capillary sequencing. Applications The Type-it Mutation Detect PCR Kit is dedicated for fast and reliable detection of mutations, such as deletions, insertions, and translocations, or for preamplification for genotyping systems (e.g., SNaPshot Kits from Applied Biosystems).
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Automated DNA fragment analysis Automated PCR setup QIAxcel System QIAgility 83 67
Type-it MM
ng 25 n 0. g 25 25 ng pg
Supplier I M
ng 25 n 0. g 25 25 ng pg
357 bp
Sensitive detection of a mutated cancer-related gene. The indicated amounts of DNA extracted from a lymphoma related cell line (Ramos) were spiked into human leukocyte DNA and the mutated Ramos target was detected together with 2 internal controls. Using the Type-it Mutation Detect PCR Kit, the mutated gene was detected even when only 25 pg of DNA was present. Electrophoresis was performed on a 1.3% agarose gel.
Product Type-it Mutation Detect PCR Kit (70) Type-it Mutation Detect PCR Kit (200)
* Includes Type-it Multiplex PCR Master Mix (with optimized MgCl2 concentration and dNTPs), containing HotStarTaq Plus DNA Polymerase, 10x CoralLoad Dye, 5x Q-Solution, and RNase-free water. Further information and online ordering: www.qiagen.com/PG/TypeitMutation Product Guide 201011 Sample & Assay Technologies 473
10.1
QIAGEN M
Supplier R
Supplier I
Supplier BIV pg
10010 1 0 10010 1
0 10010 1 0 10010 1 0
Efficient, sensitive RT-PCR. One-step RT-PCR was carried out using the indicated amounts of total RNA from HeLa cells and primers specic for -catenin amplifying a 690 bp product. All reactions were carried out following suppliers instructions. Arrow indicates primer-dimers. The QIAGEN OneStep RT-PCR Kit provided highly sensitive and specic results even with low amounts of template. M: 100 bp ladder.
Fast and easy one-tube setup One-step RT-PCR of any RNA template without optimization Unique enzyme mix for high specificity and sensitivity Optimized reverse-transcription and amplification buffer
Product description The QIAGEN One-Step RT-PCR Kit provides a blend of Sensiscript and Omniscript Reverse Transcriptases, HotStarTaq DNA Polymerase, QIAGEN OneStep RT-PCR Buffer, a dNTP mix, and Q-Solution, a novel additive that enables efficient amplification of difficult (e.g., GC-rich) templates (for more information, see page 463). The easy one-tube setup and optimized components result in highly sensitive and successful results. Applications The QIAGEN OneStep RT-PCR Kit is suitable for all standard and highly sensitive RT-PCR applications such as gene expression analysis, virus detection, and single-cell RT-PCR.
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00 ,0
00
22
22
22
22
Effective detection of viral RNA. A 336 bp fragment of F-gene mRNA was reverse-transcribed and amplied from Sendai virus RNA isolated from persistently infected Vero cells. Reactions were prepared using the QIAGEN OneStep RT-PCR Kit and the indicated number of viral genome copies. M: markers. (Data kindly provided by H. Rausch, Max Planck Institute for Biochemistry, Martinsried, Germany as part of the project Experimental control of virological work at safety levels 2 and 3 in Bavaria, supported by the Bavarian Ministry of the Environment.)
Product QIAGEN OneStep RT-PCR Kit (25) QIAGEN OneStep RT-PCR Kit (100)
copies
* Includes QIAGEN OneStep RT-PCR Enzyme Mix, 5x QIAGEN OneStep RT-PCR Buffer (contains 12.5 mM MgCl2), dNTP Mix (10 mM each dNTP), 5x Q-Solution, and RNase-free water. Further information and online ordering: www.qiagen.com/PG/OneStep
474
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10.1
Tu bu lin
TN FR 1
hT SF
Dy ne in
Highly sensitive amplification of large and rare transcripts. The QIAGEN LongRange 2Step RT-PCR Kit was used to amplify the indicated sequences from total RNA from mouse and human cell lines. The sensitivity of the kit is shown by the high yields of TNFR1 and dynein sequences, both of which are rare transcripts.
RNase H Activity
Polymerase Activity
Full length cDNA amplification due to unique RNase H activity quencher. The RNase H Activity Quencher (a component of the RT Buffer) binds to the cDNA/RNA hybridbinding site of the reverse transcriptase, preventing degradation of the RNA during synthesis. This results in reduced RNase H activity and the amplication of full-length cDNA up to 12.5 kb.
475
10.1
Product QIAGEN LongRange 2Step RT-PCR Kit (20) QIAGEN LongRange 2Step RT-PCR Kit (100)
Contents For 10 x 20 l RT reactions and 20 x 50 l PCR reactions* For 50 x 20 l RT reactions and 100 x 50 l PCR reactions*
* Includes reagents for reverse transcription and PCR. For RT step: LongRange Reverse Transcription Enzyme, buffer, dNTPs, oligo-dT, RNase inhibitor, RNase-free water. For PCR step: QIAGEN LongRange PCR Kit (see page 469).
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10.1
Omniscript RT Kit
For reverse transcription of 50 ng to 2 g RNA per reaction
1000 500
250
125
62
31
ng RNA Omniscript (QIAGEN) MMLV RNase H (Supplier I) MMLV (Supplier I) AMV RNase H (Supplier I) AMV (Supplier P)
High cDNA yields due to high-affinity enzyme Sensitive detection of as few as 10 copies of template Fast and easy procedure with no tedious pipetting steps No additional RNase H digestion step required
Product description The Omniscript RT Kit is specially designed for reverse transcription with any amount of RNA from 50 ng to 2 g per reaction. A high affinity for RNA allows Omniscript Reverse Transcriptase to provide superior performance compared with other reverse transcriptases, delivering higher sensitivity in RT-PCR, even with low-copy numbers. In addition, the combination of this enzyme with the unique reaction buffer enables read-through of templates with secondary structures. Applications The Omniscript RT Kit is the ideal choice for applications such as:
Superior sensitivity and dynamic range of Omniscript RT. Reverse transcription was carried out with different reverse transcriptases according to suppliers specications, using the indicated amounts of total RNA from HeLa cells. 1/20 of the reverse-transcription reaction was used in a 25-cycle PCR amplication with QIAGEN Taq DNA Polymerase. A 1.7 kb b-actin fragment was amplied.
Standard RT and RT-PCR Quantitative, real-time RT-PCR Primer extension and RACE analysis Synthesis of double-stranded cDNA for cloning
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Sample disruption RNA purification End-point PCR TissueLyser LT TissueLyser II RNeasy Kits HotStarTaq DNA Polymerase 41 42 409 462
Product Omniscript RT Kit (10) Omniscript RT Kit (50) Omniscript RT Kit (200)
* Includes Omniscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix (5 nM of each dNTP), and RNase-free water. Further information and online ordering: www.qiagen.com/PG/Omniscript Product Guide 201011 Sample & Assay Technologies 477
10.1
500
50
10
cells 210 bp
Sensiscript RT Kit
For reverse transcription of less than 50 ng RNA per reaction