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10

446

PCR Assay Technologies

From Sample to Result

10.1 End-point PCR and RT-PCR 10.2 Real-time PCR and RT-PCR 10.3 Cloning

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Product Guide 201011

PCR Assay Technologies

10

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

Product Guide 201011

Sample & Assay Technologies

447

PCR Assay Technologies

10

10.1 End-point PCR and RT-PCR


Selection guides
End-point PCR: applications End-point PCR: features End-point RT-PCR: applications End-point RT-PCR: features

www.qiagen.com/PG/PCR

452 453 454 454

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

Ready-to-use PCR master mixes


Standard PCR, fridge storage Standard PCR Hot-start PCR Rapid hot-start PCR TopTaq Master Mix Kit Taq PCR Master Mix Kit HotStarTaq Plus Master Mix Kit HotStarTaq Master Mix Kit QIAGEN Fast Cycling PCR Kit 456 459 461 465 466

dNTPs
PCR grade dNTP Set, PCR Grade dNTP Mix, PCR Grade 467 467

PCR kits for challenging applications


High-fidelity, hot-start PCR Long-range PCR Multiplex, hot-start PCR Template amplification for Pyrosequencing analysis Methylation-specific PCR (MSP) Methylation analysis using HRM analysis HotStar HiFidelity Polymerase Kit QIAGEN LongRange PCR Kit QIAGEN Multiplex PCR Kit
New

468 469 470 471 162 165

PyroMark PCR Kit

EpiTect MSP Kit


New

EpiTect HRM PCR Kit

448

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Product Guide 201011

PCR Assay Technologies

10

n PCR kits dedicated for genotyping applications


n Analysis of microsatellite, STR, and VNTR loci using multiplex PCR n Detection of mutations using multiplex PCR n SNP genotyping using probe-based real-time PCR with fast cycling n Detection of gene mutations and SNPs using HRM analysis Type-it Microsatellite PCR Kit Type-it Mutation Detect PCR Kit Type-it Fast SNP Probe PCR Kit
New

472 473 507 516

Type-it HRM PCR Kit

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

n DNA cleanup from PCR n Whole genome amplification

See PCR cleanup See Whole genome amplification

10.2 Real-time PCR and RT-PCR


n Selection guides

www.qiagen.com/PG/realtime

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

479 480 481 482

Product Guide 201011

Sample & Assay Technologies

449

PCR Assay Technologies

10

Real-time RT-PCR directly from cultured cells


From cells directly to cDNA From cells directly to RT-PCR results FastLane Cell cDNA Kit FastLane Cell RT-PCR Kits 483 484

Preparation of cDNA for real-time PCR


Fast cDNA synthesis with integrated genomic DNA removal cDNA synthesis from limited amounts of RNA by whole transcriptome amplification QuantiTect Reverse Transcription Kit 486

QuantiTect Whole Transcriptome Kit

445

Real-time PCR and RT-PCR using SYBR Green I


Genomewide, predesigned primer sets Ultrafast cycling on Rotor-Gene cyclers Fast cycling Standard cycling with optional UNG pretreatment microRNA and mRNA detection QuantiTect Primer Assays
New New

487 487 489 491 494 558

QuantiTect Primer Assay Plates Rotor-Gene SYBR Green Kits

QuantiFast SYBR Green Kits QuantiTect SYBR Green Kits miScript PCR System

Real-time PCR and RT-PCR using sequence-specific probes


Ultrafast cycling on Rotor-Gene cyclers Fast cycling Standard cycling with optional UNG pretreatment Viral DNA and RNA detection SNP genotyping Methylation analysis
New

Rotor-Gene Probe Kits

497 499 502 505 507 163 164

QuantiFast Probe Kits QuantiTect Probe Kits QuantiTect Virus Kits Type-it Fast SNP Probe PCR Kit EpiTect MethyLight PCR Kits EpiTect MethyLight Assays

Multiplex, real-time PCR and RT-PCR using sequence-specific probes


Ultrafast cycling on Rotor-Gene cyclers Fast cycling
New

Rotor-Gene Multiplex Kits QuantiFast Multiplex RT-PCR Kits

508 510 510

QuantiFast Multiplex PCR Kits


New

450

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Product Guide 201011

PCR Assay Technologies

10

n Standard cycling with optional UNG pretreatment n Viral DNA and RNA detection

QuantiTect Multiplex Kits QuantiTect Virus Kits

513 505

n HRM (high-resolution melt) analysis


n Detection of gene mutations and SNPs n Methylation analysis
New New

Type-it HRM PCR Kit EpiTect HRM PCR Kit

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

www.qiagen.com/PG/cloning 362

QIAGEN PCR Cloningplus Kit QIAGEN PCR Cloning Kit

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.

Product Guide 201011

Sample & Assay Technologies

451

PCR Assay Technologies

10.1

End-point PCR: applications


Fa st E C Ty N M ycl in pe u g lti -it P pl M Ty ex CR i cr pe K P os CR it -it M ate K Q l u it li IA G tatio te P E CR Ho N L n D Ki o et tS ta ngR ect t r a P Q Hi ng CR IA Fi e d G PC Kit EN eli R t Q Ki Ta y P IA t o q G EN DN lym Q e A Ta r IA a Po q se G ly P E Ki m To N T CR t e C a r pT aq q P ore ase CR K D To M it pT NA aq Po ast Ho Ma lym er M ste er tS i ta as x K r it Ho rTaq Mix e tS DN Ki ta t r A Ho Taq Po tS ly M ta m a s Ho rTaq ter era s M tS P ix e ta lu s r K T Ep DN a it iTe q P A lu ct s M Pol M SP as ym e Ki te r M ras t ix e Ki t IA G
PCR
466 470 472 473 469 468 457 457 459 455 456

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.

IA

EN


462


465


460


461 162

452

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Product Guide 201011

End-point PCR and RT-PCR

10.1

End-point PCR: features


Fa st E C Ty N M yc li pe u lti ng -it PC pl M Ty e pe icro x P R K it* CR -it s M ate Ki Q lli ut t IA t a e G tio PC E R Ho N L n D Ki o e tS t ta ngR ect t r a P Q Hi ng CR IA Fi e d G PC Kit EN eli R t Q Ki Ta y P IA t o q G EN DN lym Q e A Ta IA Po rase q G ly P E Ki m To N T CR Co era t aq pT aq se PC re K D R To M it pT NA aq Po ast Ho Ma lym er M ste er tS i ta as x K r it Ho rTaq Mix e tS DN Ki ta t r A Ho Taq Po tS ta Mas lym r Ho Taq ter era s M tS Pl ix e ta u Ki Ep rTaq s D t N iTe A Pl ct us Po M SP Ma lym st e Ki t er M ras ix e Ki t IA G
PCR n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n
466 470 472 473 469 468 457 457 459 455

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

IA

EN

n n
456 462

465

460

461

162

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.

Product Guide 201011

Sample & Assay Technologies

453

PCR Assay Technologies

10.1

End-point RT-PCR: applications


Lo ng Ra n m O ni ne ge s 2 St ep Ste Se crip p tR ns R RT isc T T rip Kit -PC -PC R R tR Ki Ki T t t Ki t G

475 474 477 478

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

End-point RT-PCR: features

Benefits PCR and RT-PCR performance

Advantages Hot-start (15 min) Maximal specificity Maximal sensitivity Q-Solution

Ease of use and convenience

Direct UA/TA cloning Complete kit format including nucleotides Fast and easy procedure

Page : Recommended product.

475

(All products shown provide reliable results without the need for optimization of PCR parameters.) * Amplification product size: 12.5 kb.

Amplification product size: 5 kb. Amplification product size: 4 kb.

454

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G EN Q IA Lo G EN ngR an O m O ni ne ge sc 2S S te ri Se t ns pt R p R ep R isc T T T rip Kit PC -PC R R tR K T it Kit* Ki t


RT-PCR
474

* Including gene expression analysis.

EN

IA

477

478

Product Guide 201011

End-point PCR and RT-PCR

10.1

TopTaq DNA Polymerase


For highly reliable end-point PCR applications with unrivaled ease-of-use

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

Product TopTaq DNA Polymerase (250 U)*

Contents For 200 x 50 l reactions

Cat. no. 200203 200205 200207

TopTaq DNA Polymerase (1000 U)* TopTaq DNA Polymerase (5000 U)*

For 800 x 50 l reactions For 4000 x 50 l reactions

* 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

Product Guide 201011

Sample & Assay Technologies

455

PCR Assay Technologies

10.1

A
M 20C 4C 25C M

TopTaq Master Mix Kit


For highly reliable end-point PCR with unrivaled ease-of-use

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.

Related products
Automated PCR setup Automated DNA fragment analysis QIAgility QIAxcel System 67 83

Product TopTaq Master Mix Kit (250 U)

Contents For 200 x 50 l reactions*

Cat. no. 200403

* 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

456

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Product Guide 201011

End-point PCR and RT-PCR

10.1

Taq DNA Polymerase and Taq PCR Core Kit


For standard and specialized PCR applications

M A07 A07

A A08 A09

B A10 A11

C A12

M A08 3000.0 603.0 194.0 15.0

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:

1078.0 310.0 118.0

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.

Product Guide 201011

Sample & Assay Technologies

457

PCR Assay Technologies

10.1

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.

Contents For 200 x 50 l reactions

Cat. no. 201203 201205 201207

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

201209 201223 201225

For 800 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).

Further information and online ordering: www.qiagen.com/PG/TaqDNA

458

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Product Guide 201011

End-point PCR and RT-PCR

10.1

Taq PCR Master Mix Kit


Premixed solution for convenient PCR setup

M C

10

20

50

copies

Easy reaction setup Fewer pipetting steps Minimal optimization


Product description Taq PCR Master Mix contains Taq 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, increasing throughput and reproducibility. Applications The Taq PCR Master Mix Kit is suitable for standard and specialized applications, including:
188 bp

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)

Contents For 200 x 50 l reactions* For 800 x 50 l reactions*

Cat. no. 201443 201445

* 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

Product Guide 201011

Sample & Assay Technologies

459

PCR Assay Technologies

10.1

H ot St ar Ta q H ot St ar Ta q Su pp lie rI Su pp lie rA ll Su pp lie rR D Po N lym A er as e

Pl

us q Ta

HotStarTaq Plus DNA Polymerase


For fast and highly specific amplification in all applications

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).
Related products
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.

Product HotStarTaq Plus DNA Polymerase (250 U)*

Contents For 200 x 50 l reactions


Cat. no. 203603 203605 203607 203609

HotStarTaq Plus DNA Polymerase (1000 U)* HotStarTaq Plus DNA Polymerase (5000 U)*

For 800 x 50 l reactions

For 4000 x 50 l reactions

HotStarTaq Plus DNA Polymerase (25,000 U)*

For 20,000 x 50 l reactions

* 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

460

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Product Guide 201011

End-point PCR and RT-PCR

10.1

HotStarTaq Plus Master Mix Kit


For fast and highly specific amplification in all applications

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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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

Cat. no. 203643 203645 203646

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.

Further information and online ordering: www.qiagen.com/PG/HotStarTaqPlusMasterMix

Product Guide 201011

Sample & Assay Technologies

461

PCR Assay Technologies

10.1

Reaction setup at room temperature

HotStarTaq DNA Polymerase


For highly specific amplification in any PCR application

Distribute

Highly specific PCR Reduced nonspecific amplification Easy handling and room-temperature setup

Add primers and template

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

3000.0 1200.0 500.0 300.0

5000.0 2000.0 600.0 400.0 200.0

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.

462

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Product Guide 201011

End-point PCR and RT-PCR

10.1

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

Product Guide 201011

Sample & Assay Technologies

463

PCR Assay Technologies

10.1

Additional kits for dedicated applications


Special feature Parallel amplification of multiple products Description Multiplex PCR Recommended kit Page 473 472 470 516 507 466 465 460 461 468 469 162 165 474 475 455 456 457 457 459

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.

Contents For 200 x 50 l reactions

Cat. no. 203203 203205 203207

For 800 x 50 l reactions For 4000 x 50 l reactions For 20,000 x 50 l reactions

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.

Further information and online ordering: www.qiagen.com/PG/HotStarTaqDNA

464

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Product Guide 201011

End-point PCR and RT-PCR

10.1

HotStarTaq Master Mix Kit


Premixed solution for high PCR specificity

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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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

Cat. no. 203443 203445 203446

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.

Further information and online ordering: www.qiagen.com/PG/HotStarTaqMasterMix

Product Guide 201011

Sample & Assay Technologies

465

PCR Assay Technologies

10.1

Significant Time Savings


Fast Cycling Procedure Enzyme activation Standard Cycling Procedure Enzyme activation

QIAGEN Fast Cycling PCR Kit


For ultrafast and specific PCR on any thermal cycler

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)

Contents For 200 x 20 l reactions For 1000 x 20 l reactions

Cat. no. 203743 203745

* 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

466

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Product Guide 201011

End-point PCR and RT-PCR

10.1

dNTP Set, PCR Grade and dNTP Mix, PCR Grade


For sensitive and reproducible PCR and RT-PCR

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.

Ideal for all PCR and RT-PCR applications


Sample type Application DNA Standard PCR High-fidelity PCR Ultrafast PCR Long-range PCR Multiplex PCR Genotyping Single-cell PCR Methylation-specific PCR (MSP) Nested PCR RNA/ cDNA Two-step RT-PCR Multiplex, two-step PT-PCR Single-cell, two-step PT-PCR dNTP Mix, PCR Grade can be used in a variety of sensitive PCR techniques.

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

Cat. no. 201900 201901 201912 201913

For 2500 x 50 l reactions

Separate aliquots of 100 mM each dATP, dCTP, dGTP, and dTTP.

Further information and online ordering: www.qiagen.com/PG/dNTPSet

Product Guide 201011

Sample & Assay Technologies

467

PCR Assay Technologies

10.1

Q IA G E Su N pp li Su er S pp lie r Su pp I * lie rI Su pp * lie rR

HotStar HiFidelity Polymerase Kit


For highly sensitive and reliable high-fidelity hot-start PCR

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.

Cloning efficiency (%)

100 80 60 40 20 0 TA cloning UA cloning

Taq DNA Polymerase HotStar HiFidelity

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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Product HotStar HiFidelity Polymerase Kit (100 U) HotStar HiFidelity Polymerase Kit (1000 U)

Contents For 40 x 50 l reactions* For 400 x 50 l reactions*

Cat. no. 202602 202605

* 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

468

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Product Guide 201011

End-point PCR and RT-PCR

10.1

For sensitive and accurate long-range PCR

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)

Contents For 20 x 50 l reactions* For 100 x 50 l reactions* For 250 x 50 l reactions*

* 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

QIAGEN LongRange PCR Kit

27 kb 24 kb

Cat. no. 206401 206402 206403

PCR Assay Technologies

10.1

Supplier I M 1.5 2.5 3.5

QIAGEN Std mM Mg2+

QIAGEN Multiplex PCR Kit


For highly specific and sensitive multiplex PCR without optimization

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)

Contents For 100 x 50 l multiplex PCRs* For 1000 x 50 l multiplex PCRs*

Cat. no. 206143 206145

* 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.

Master mix supplied in single tube.

Further information and online ordering: www.qiagen.com/PG/QIAGENMultiplexPCR

470

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Product Guide 201011

End-point PCR and RT-PCR

10.1

New

PyroMark PCR Kit


125 100 75 50 25 0

PyroMark PCR Kit


B7: TACTCRTAAATACRACRCTTATATTCTTAAAAAAAATTTTAC 50% 58% 45%

For PCR amplification of template DNA optimized for Pyrosequencing analysis

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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72

Product PyroMark PCR Kit (200) PyroMark PCR Kit (800)

Contents For 200 reactions* For 800 reactions*

Cat. no. 978703 978705

* 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

PCR Assay Technologies

10.1

FAM Channel
5000 4000 3000 2000 1000 0 80

Type-it Microsatellite PCR Kit


For fast and reliable multiplex PCR analysis of microsatellite loci


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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Product Type-it Microsatellite PCR Kit (70) Type-it Microsatellite PCR Kit (200)

Contents For 70 x 25 l reactions* For 200 x 25 l reactions*

Cat. no. 206241 206243

* 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

472

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Product Guide 201011

End-point PCR and RT-PCR

10.1

Type-it Mutation Detect PCR Kit


For accurate and reliable multiplex PCR analysis of mutations

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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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)

Contents For 70 x 25 l reactions* For 200 x 25 l reactions*

Cat. no. 206341 206343

* 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

PCR Assay Technologies

10.1

QIAGEN M

Supplier R

Supplier I

Supplier BIV pg

10010 1 0 10010 1

0 10010 1 0 10010 1 0

QIAGEN OneStep RT-PCR Kit


For highly sensitive and successful one-step RT-PCR


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

Contents For 25 x 50 l reactions* For 100 x 50 l reactions*

Cat. no. 210210 210212

* 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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Product Guide 201011

End-point PCR and RT-PCR

10.1

Tu bu lin

TN FR 1

For sensitive and accurate long-range two-step RT-PCR

M 12.3 kb 5.3 kb 13 kb 500 bp

hT SF

Dy ne in

QIAGEN LongRange 2Step RT-PCR Kit


Dedicated solution for amplification of long transcripts High-yield amplification of up to 12.5 kb cDNA Low error rates and extended read length Minimal PCR optimization due to unique buffer system Amplification of low-abundance and GC-rich templates
Product description The QIAGEN LongRange 2Step RT-PCR Kit combines the features and benefits of the QIAGEN LongRange PCR Kit with a high-yield reverse-transcription step. The recombinant homodimeric viral reverse transcriptase (AMV) provides highly sensitive, full-length cDNA synthesis up to 12.5 kb. The special buffer composition, which includes a novel RNase H activity quencher (see figure), allows reverse transcription with no optimization required. In combination with the components for long-range PCR, 3 powerful enzymes enable long and accurate two-step amplification, even for low-copy targets. The kit comes complete and ready to use with all components necessary for successful results. Preoptimized buffers reduce the need for lengthy protocol optimization. Applications The QIAGEN LongRange 2Step RT-PCR Kit is suitable for dedicated long-range two-step RT-PCR applications such as cloning and gene expression analysis. The high sensitivity and accuracy make this kit highly suited for amplifying low-abundance targets.
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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

RNase H Activity Quencher X

Polymerase Activity RNA


X

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.

Product Guide 201011

Sample & Assay Technologies

475

PCR Assay Technologies

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*

Cat. no. 205920 205922

* 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).

Further information and online ordering: www.qiagen.com/PG/LongRange2Step

476

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Product Guide 201011

End-point PCR and RT-PCR

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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Product Omniscript RT Kit (10) Omniscript RT Kit (50) Omniscript RT Kit (200)

Contents For 10 x 20 l reactions* For 50 x 20 l reactions* For 200 x 20 l reactions*

Cat. no. 205110 205111 205113

* 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

PCR Assay Technologies

10.1

500

50

10

cells 210 bp

Sensiscript RT Kit
For reverse transcription of less than 50 ng RNA per reaction

-Actin

NF- B

460 bp

Inositol receptor

240 bp

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
Dystrophin
360 bp

RT-PCR with RNA corresponding to 1 cell. Total RNA was puried from 10 to 5000 HeLa cells using RNeasy technology. 1/10 of each eluate (corresponding to RNA from 1 to 500 cells) was used for reverse transcription with Sensiscript Reverse Transcriptase. 1/2 of each RT reaction was then used in a 40-cycle PCR with QIAGEN Taq DNA Polymerase and primers specic for genes encoding -actin, NF-B, inositol-1,4, 5-triphosphate receptor, or dystrophin. Sizes of the amplicons are as indicated.

The Sensiscript RT Kit is ideally suited for highly sensitive applications using very small amounts of RNA (less than 50 ng), such as single-cell RT-PCR and analysis of biopsies and LMD samples. Sensiscript Reverse Transcriptase allows highly efficient RT-PCR over a wide dynamic range and extremely sensitive RT-PCR with very small RNA amounts due to its high affinity for RNA. In addition, the combination of this enzyme with the unique reaction buffer enables read-through of templates with secondary structures. Applications The Sensiscript RT Kit is well suited for applications such as:

Standard RT and RT-PCR Quantitative, real-time RT-PCR Differential display RT-PCR Primer extension and RACE analysis Synthesis of double-stranded cDNA for cloning

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Product Sensiscript RT Kit (50) Sensiscript RT Kit (200)

Contents For 50 x 20 l reactions* For 200 x 20 l reactions*

Cat. no. 205211 205213

* Includes Sensiscript 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/Sensiscript

478

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

Real-time PCR and RT-PCR: ultrafast, precise quantification on Rotor-Gene cyclers


Ro to rRo Gen e to SY rG en BR Ro G e to re SY rG BR en e Ro P ne G to re CR Pr rKi en ob G t e Ro R e PC T-P to ne P C rR ro R G Ki be Ki e Ro t t to ne M RT r-P ul G tip CR en Ki e le t x M PC ul tip R le Ki x RT t -P CR Ki t
Detection SYBR Green Template Genomic DNA cDNA RNA Cells Probe Genomic DNA cDNA RNA Cells Multiplex Genomic DNA cDNA RNA Cells Page n: Recommended product. * Rotor-Gene Kits are supplied with a master mix containing dATP, dCTP, dGTP, and dTTP. If UNG treatment needs to be carried out, use QuantiTect Kits (page 481).

Procedure PCR* Two-step RT-PCR* One-step RT-PCR* Two-step RT-PCR* PCR* Two-step RT-PCR* One-step RT-PCR* Two-step RT-PCR* PCR* Two-step RT-PCR* One-step RT-PCR* Two-step RT-PCR*
489

Real-time PCR/RT-PCR n n n n n n n n n n n n
489 497 497 508 508

Kit can be used in combination with QuantiTect Primer Assays, which are genomewide primer sets designed for highly specific quantification (page 487). For fast cDNA synthesis with genomic DNA removal, use the QuantiTect Reverse Transcription Kit (page 486). To prepare unlimited amounts of cDNA from precious RNA samples, use the QuantiTect Whole Transcriptome Kit (page 445). Requires additional use of the FastLane Cell cDNA Kit (page 483), which prepares cDNA directly from cells without RNA purification.

Product Guide 201011

Sample & Assay Technologies

479

PCR Assay Technologies

10.2

Real-time PCR and RT-PCR: fast cycling


iF a ua st S YB nt iF as R G Q ua tS re nt iF YBR en P as Q CR G ua tP re Ki nt en iF rob t a Ty R e PC T-P pe st P C -it R ro R Ki be F Ki Q t ua ast PC t S nt R N iF + P RO a Q ua st P Pro be X V ro nt ia iF b P e as Q lK RT CR ua tP it K nt P CR it iF rob as Q e ua t M RT Kit -P nt u CR iF as ltip Q ua t M lex +R O nt P u iF CR X V as ltip Q ia ua Ki t M lex lK nt PC t ul it i tip R Ty Fas +R t M lex pe -it Ki RT ul t -P Ep HR tipl ex CR M iTe Ki PC ct RT t R HR Ki PCR M +R PC t R Ki Ki t t Q
Real-time PCR/RT-PCR
491 491 499 499 507 499 499 510 510 510 510 516 165

Detection SYBR Green

Template Genomic DNA cDNA RNA

Procedure PCR* Two-step RT-PCR* One-step RT-PCR* PCR*

ROX dye In master mix In master mix In master mix In master mix In separate tube

Probe

Genomic DNA cDNA

Two-step RT-PCR* One-step RT-PCR* PCR*

In master mix In separate tube In master mix In separate tube In master mix In separate tube

RNA

Multiplex

Genomic DNA cDNA

Two-step RT-PCR* One-step RT-PCR* PCR* PCR*

In master mix In separate tube In master mix In separate tube Not included Not included

RNA

HRM

Genomic DNA Bisulfiteconverted DNA

Page : Recommended product.

* Kits are supplied with a master mix containing dATP, dCTP, dGTP, and dTTP. If UNG treatment needs to be carried out, use QuantiTect Kits (page 481).

Kit can be used in combination with QuantiTect Primer Assays, which are genomewide primer sets designed for highly specific quantification (page 487). For fast cDNA synthesis with genomic DNA removal, use the QuantiTect Reverse Transcription Kit (page 486). To prepare unlimited amounts of cDNA from precious RNA samples, use the QuantiTect Whole Transcriptome Kit (page 445). For SNP genotyping using TaqMan MGB probes.

480

ua nt

www.qiagen.com

Product Guide 201011

Real-time PCR and RT-PCR

10.2

Real-time PCR and RT-PCR: standard cycling


iTe ua ct S nt YB iTe R Q G ua ct S YB ree nt n iTe R PC Q ua ct P Gr ee R K ro nt n i iTe b RT t e Q P -P ua ct P CR ro CR nt Ki i be Ki Q Tect t RT t ua M PC nt u lti iTe R p Q ua ct M lex Kit nt iTe ulti PCR Q p Ki ua ct M lex t nt PC iTe ulti R Q p N ua ct M lex oR nt RT iTe ulti -P OX Q pl c C t Ki ua ex R t nt Vir us RT- Kit i PC Ep Tect Ki R iTe t Vi N ru ct R Ki Ep Me s + t R th iTe O yL ct X i g V M +R h i O ethy t PC al K X it R Vi Lig Ki ht al t P Ki CR t Q
Real-time PCR/RT-PCR n

Detection Template SYBR Green Genomic DNA cDNA RNA Probe Genomic DNA cDNA RNA

Procedure ROX dye PCR* Two-step RT-PCR* In master mix In master mix

n n n n n n n n n n n n n n n n n
494 494 502 502 513 513 513 513 505 505 163 163

One-step In master mix RT-PCR* PCR* Two-step RT-PCR* In master mix In master mix

One-step In master mix RT-PCR*

Viral DNA PCR and In master mix and RNA one-step In separate tube RT-PCR Bisulfiteconverted DNA Multiplex Genomic DNA cDNA PCR In master mix In separate tube PCR* In master mix Not included Two-step RT-PCR* In master mix Not included

RNA

One-step In master mix RT-PCR* Not included

Viral DNA PCR and In master mix and RNA one-step In separate tube RT-PCR Page

n: Recommended product. * UNG treatment possible (QuantiTect SYBR Green, Probe, and Multiplex Kits are supplied with a master mix containing dATP, dCTP, dGTP, and dTTP/dUTP). UNG treatment not possible (QuantiTect Virus and EpiTect Kits are supplied with a master mix containing dATP, dCTP, dGTP, and dTTP). Kit available with or without UNG. Kit can be used in combination with QuantiTect Primer Assays, which are genomewide primer sets designed for highly specific quantification (see page 487). For fast cDNA synthesis with genomic DNA removal, use the QuantiTect Reverse Transcription Kit (page 486). To prepare unlimited amounts of cDNA from precious RNA samples, use the QuantiTect Whole Transcriptome Kit (page 445).

Product Guide 201011

ua nt

Sample & Assay Technologies

481

PCR Assay Technologies

10.2

Real-time RT-PCR: direct from cell lysates without RNA purification


iTe ua ct S YB nt iTe R Q ua ct P Gr ee ro nt n iTe be P Q ua ct M PC CR R Ki nt u Ki iTe lti t Q p t ua ct M lex nt P u C i l Q Fas tiple R K ua tS i YB x PC t nt iF as R G R N Q ua tP re oR nt iF rob en P OX as Q e C K ua PC tP R Ki it nt R iF rob t K as i Q e ua t M PC t nt R ul iF + tip R a Fa stL st M lex OX an Vi PC ul a R Fa e C tipl Ki l Ki ex el stL t an l SY PC t e R B Fa R + stL Cel l P Gre R K an it en ro e Fa Ki stL Cel be t K l an i M e ul t Ce tip ll le x M Ki ul tip t le x N R Ki t Q
Complete kits
494 502 513 513 491 499 499 510 510 484 484 484 484

Detection Procedure Cycling SYBR Green Two-step RT-PCR* Standard Fast One-step RT-PCR Probe Two-step RT-PCR*

ROX dye In master mix In master mix

Standard In master mix Standard In master mix Fast In master mix In separate tube

One-step RT-PCR Multiplex Two-step RT-PCR*

Standard In master mix Standard In master mix Not included Fast In master mix In separate tube

One-step RT-PCR

Standard In master mix Not included

Page : Recommended product.

* cDNA synthesis direct from cell lysates, followed by real-time PCR. See also the selection guide on page 479.

Real-time RT-PCR direct from cell lysates. UNG treatment possible with standard-cycling kits (kits are supplied with a master mix containing dATP, dCTP, dGTP, and dTTP/dUTP). UNG treatment not possible with fast-cycling kits (kits are supplied with a master mix containing dATP, dCTP, dGTP, and dTTP). Kit can be used in combination with QuantiTect Primer Assays, which are genomewide primer sets designed for highly specific quantification (see page 487).

482

ua nt

With FastLane Cell cDNA Kit (page 483)

www.qiagen.com

Product Guide 201011

Real-time PCR and RT-PCR

10.2

FastLane Cell cDNA Kit


For high-speed preparation of cDNA without RNA purification for use in real-time RT-PCR

A
Relative expression of CDC2 (%) 160 120 80 40 0 S1 S2 S3 NS UT

From cells to cDNA in only 4 steps and under 45 minutes Easy parallel processing of several samples Sensitive detection of even low-abundance transcripts Integrated gDNA removal allows detection of RNA only No need to design RNA-specific primers or probes
Product description

Relative expression of CDC2 (%)

The kit provides a fast and simple procedure for preparing first-strand cDNA directly from cultured cells without RNA purification. The kit is supplied with wash and lysis buffers for preparing lysates and stabilizing RNA, with gDNA Wipeout Buffer for eliminating genomic DNA contamination, and with all reaction components for fast and efficient cDNA synthesis. Applications cDNA synthesized by the FastLane Cell cDNA Kit gives highly sensitive and reproducible results in real-time two-step RT-PCR. The kit is ideal for experiments which require a snapshot of individual transcript levels, such as:

B
160 120 80 40 0 S1 S2 S3 NS UT

Validation of siRNA-mediated gene knockdown Evaluation of drug effects Detection of gene regulation
Related products
Gene silencing Ultrafast real-time PCR Fast real-time PCR Real-time PCR with UNG pretreatment FlexiTube siRNA Rotor-Gene Kits QuantiFast Kits QuantiTect Kits 543 479 480 481

CDC2 knockdown in cultured cells. A MCF-7 cells were transfected with various CDC2 siRNAs (S1, S2, or S3) or with nonsilencing siRNA (NS). After 48 hours, the relative expression of CDC2 was determined by real-time RT-PCR. The relative expression of CDC2 in untreated cells (UT) was set to 100%. cDNA was prepared using the FastLane Cell cDNA Kit. B The experiment was repeated on HeLa cells.

Product FastLane Cell cDNA Kit (50)

Contents For 50 x 20 l reactions*

Cat. no. 215011

* Includes Buffer FCW, Buffer FCP, gDNA Wipeout Buffer, Quantiscript Reverse Transcriptase, 5x Quantiscript RT Buffer (with dNTPs), RT Primer Mix, and RNase-free water. The kit contains sufcient reagent for preparing cDNA from 2 x 24-well cell culture plates or 1 x 48-well cell culture plate. cDNA preparation from 1 x 96-well cell culture plate is possible with the additional purchase of a QuantiTect Reverse Transcription Kit (50) (page 486). Further information and online ordering: www.qiagen.com/PG/FastLaneCellcDNA Product Guide 201011 Sample & Assay Technologies 483

PCR Assay Technologies

10.2

FastLane Cell RT-PCR Kits


For real-time one-step RT-PCR analysis directly from cultured cells without RNA purification

No RNA purification required, significantly saving time Just 3 steps from cells to real-time RT-PCR High-throughput analysis of 96- and 384-well plates Unique gDNA Wipeout Buffer enables detection of RNA only Immediate startup using optimized reagents and protocols

Product description FastLane Cell RT-PCR Kits provide a set of buffers for preparing FastLane lysates from cultured cells.* In addition to lysing cells, the buffers also stabilize cellular RNA and eliminate genomic DNA. FastLane lysates are used directly in real-time one-step RT-PCR. A variety of detection formats are available: detection with SYBR Green or sequence-specific probes, or multiplex detection with sequence-specific probes (with or without ROX passive reference dye). Applications The kits are ideal for experiments which require a snapshot of individual transcript levels, such as:

Validation of siRNA-mediated gene knockdown Evaluation of drug effects Detection of gene regulation
* For primary cells, please contact QIAGEN Technical Services.

n = 384 Mean C T = 25.24 CV = 1.97%

Highly reproducible analysis of a 384-well plate. Real-time one-step RT-PCR analysis of the expression of NFB1 (nuclear transcription factor) using the FastLane Cell Probe Kit gave reproducible CT values from a 384-well plate of HeLa cells.

484

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

HeLa

HepG2

Cos

Sensitive detection without RNA purification. Two low-abundance transcripts, INPP5D (a phosphatase) and SRF (serum response factor), were quantied by real-time one-step RT-PCR. The FastLane Cell SYBR Green Kit provided CT values comparable to those achieved with the RNeasy Mini Kit and QuantiTect SYBR Green RT-PCR Kit.

Reliable validation of gene silencing. In a 96-well plate of HCT116 cells, 5 wells were transfected with lamin A/C siRNA (Lamin) or nonsilencing siRNA (NS). Cells were analyzed by multiplex, real-time RT-PCR using the FastLane Cell Multiplex Kit and TaqMan assays for lamin A/C (structural protein) and 18S rRNA (endogenous control). (Data kindly provided by Angela Quinn, Genzyme Corporation, USA.)

Related products
Gene silencing Transfection Gene expression assays FlexiTube siRNA FlexiPlate siRNA HiPerFect Transfection Reagent QuantiTect Primer Assays 543 547 569 487

Product FastLane Cell SYBR Green Kit (200)* FastLane Cell Probe Kit (200)

Contents For 200 x 50 l reactions


Cat. no. 216213 216413 216513 216713

For 200 x 50 l reactions For 200 x 50 l reactions For 200 x 50 l reactions

FastLane Cell Multiplex Kit (200) FastLane Cell Multiplex NR Kit (200)
* For real-time one-step RT-PCR using SYBR Green I.

Includes FastLane Cell One-Step Buffer Set, RT-PCR master mix, RT mix, and RNase-free water. The kit contains sufcient reagent for real-time one-step RT-PCR from 2 x 96-well cell culture plates (50 l PCR volume) or 1 x 384-well cell culture plate (25 l PCR volume). For real-time one-step RT-PCR using sequence-specic probes. For multiplex, real-time one-step RT-PCR. Recommended for cyclers that require ROX dye for uorescence normalization (e.g., all instruments from Applied Biosystems). For multiplex, real-time one-step RT-PCR. Recommended for cyclers that do not require ROX dye for uorescence normalization (e.g., Rotor-Gene cyclers and instruments from Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene).

Further information and online ordering: www.qiagen.com/PG/FastLaneCellRTPCR Product Guide 201011 Sample & Assay Technologies 485

PCR Assay Technologies

10.2

290 240 190

QIAGEN Supplier I Supplier AII

QuantiTect Reverse Transcription Kit


For fast cDNA synthesis for sensitive real-time two-step RT-PCR

140 90 40 10 10 20 30 40 50

cDNA synthesis and gDNA removal in only 20 minutes No need to design RNA-specific primers or probes High cDNA yields even from low-abundance transcripts cDNA synthesis from 5' and 3' regions of transcripts

Rn

Product description The unique QuantiTect Reverse Transcription Kit provides a fast and convenient procedure for cDNA synthesis with integrated genomic DNA removal. Genomic DNA contamination in RNA samples is effectively eliminated by gDNA Wipeout Buffer. Fast and efficient reverse transcription is provided by Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, and unique RT Primer Mix. The synthesized cDNA is optimized for use in real-time PCR, allowing reliable quantification of targets from all regions of mRNA transcripts. Applications The QuantiTect Reverse Transcription Kit is specifically designed for use in real-time two-step RT-PCR, and provides high cDNA yields for sensitive quantification of even low-abundance transcripts. Even RNA from small amounts of cells and tissues (down to 10 pg RNA) is suitable as starting material.

Cycle Higher sensitivity in real-time two-step RT-PCR. Real-time two-step RT-PCR analysis of TGFB2 (low expression) was carried out. cDNA was synthesized from 1 g human blood RNA using the QuantiTect Reverse Transcription Kit, a kit from Supplier AII, or a kit from Supplier I. Real-time PCR was performed in duplicate on the ABI PRISM 7900 using the QuantiTect Probe PCR Kit and a gene expression assay for TGFB2. The CT values were lowest with the QuantiTect Reverse Transcription Kit, demonstrating that even low-abundance transcripts can be efciently reverse transcribed and sensitively detected in real-time PCR.

Related products
RNA purification Ultrafast real-time PCR Fast real-time PCR Real-time PCR with UNG pretreatment RNeasy Kits Rotor-Gene Kits QuantiFast Kits QuantiTect Kits 409 479 480 481

Product QuantiTect Reverse Transcription Kit (10) QuantiTect Reverse Transcription Kit (50) QuantiTect Reverse Transcription Kit (200)

Contents For 10 x 20 l reactions* For 50 x 20 l reactions* For 200 x 20 l reactions*

Cat. no. 205310 205311 205313

* Includes gDNA Wipeout Buffer, Quantiscript Reverse Transcriptase, 5x Quantiscript RT Buffer (with dNTPs), RT Primer Mix, and RNasefree water. Further information and online ordering: www.qiagen.com/PG/QuantiTectRT

486

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

QuantiTect Primer Assays


For use in real-time RT-PCR with SYBR Green detection

A
800 700 600 500 400 300 200 100 0 -100 100 ng 10 ng 1 ng 0.1 ng

Guaranteed results with genomewide, predesigned primers ~100% PCR efficiencies for reliable relative quantification High sensitivity and specificity Accurate quantification over a wide linear range Time and cost savings with SYBR Green detection
Product description QuantiTect Primer Assays are genomewide, bioinformatically validated primer sets for use in SYBR Green-based real-time RT-PCR on any cycler. Assays are available for all genes from human, rat, mouse, and many other species. Each assay for a specific gene is supplied as a lyophilized mix of forward and reverse primers that can be easily reconstituted to obtain a 10x assay solution (reaction components for real-time RT-PCR need to be ordered separately). Assays are available either in single tubes or in 96- or 384-well plates. When used in combination with QuantiTect, QuantiFast , Rotor-Gene, or FastLane Kits for SYBR Green detection, QuantiTect Primer Assays guarantee highly specific and sensitive results in real-time RT-PCR comparable to probe-based detection. QuantiTect Primer Assays are designed to detect RNA sequences only where possible. QuantiTect Primer Assays can be searched for and ordered online at the easy-to-use QIAGEN GeneGlobe Web portal (page 524). Simply enter a few details at the GeneGlobe search page to find the assay(s) for individual target genes. Depending on the gene, assays for specific transcript variants are also available. Applications QuantiTect Primer Assays are ideal for gene expression analysis applications, such as validation of siRNA-mediated gene knockdown or validation of microarray results.

PCR base line subtracted CF RFU

10 15 20 25 Cycle

30 35 40 45

B
100 80 d(RFU) / dT 60 40 20 0 -20 66 69 72 75 78 81 84 87 90

Temperature (C)

Reproducible real-time RT-PCR on the iCycler iQ. A Different amounts of leukocyte cDNA were analyzed in duplicate using the QuantiTect Primer Assay for IL8 (a chemokine) and QuantiFast SYBR Green PCR Kit. No template control (NTC) reactions were also performed (shown by the at plots). B Melting curve analysis, demonstrating high PCR specicity.

Product Guide 201011

Sample & Assay Technologies

487

PCR Assay Technologies

10.2

SYBR Green-based assay (QIAGEN) 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 5 MAPK1, CT = 25.1 IL6, CT = 30.7

Probe-based assay (Supplier AII) 2.5 MAPK1, CT = 29.5 IL6, CT = 33.7

Derivative reporter (-Rn)

4.0 3.0 2.0 1.0 0

Rn

Rn
95

MAPK1 IL6

2.0 1.5 1.0 0.5

10 15

20 25 30 35 40 Cycle

60 65 70 75 80 85 90 Temperature (C)

0 5 10 15 20 25 30 35 40 Cycle

Superior sensitivity in real-time RT-PCR on the StepOnePlus. MAPK1 (a protein kinase) and IL6 (a cytokine) in human leukocyte cDNA (1 ng) were quantied in duplicate using A QuantiTect Primer Assays and the QuantiTect SYBR Green PCR Kit, or B probe-based assays and a real-time PCR kit from Supplier AII. QuantiTect Primer Assays provided greater sensitivity than the probe-based assays, as demonstrated by the lower CT values, as well as high specicity, as indicated by the single peaks in melting curve analysis.

Related products
Reaction setup Real-time cycler cDNA synthesis QIAgility Rotor-Gene Q QuantiTect Reverse Transcription Kit QuantiTect Whole Transcriptome Kit FastLane Cell cDNA Kit Real-time RT-PCR Rotor-Gene SYBR Green Kits QuantiFast SYBR Green Kits QuantiTect SYBR Green Kits FastLane Cell SYBR Green Kit 67 78 486 445 483 489 491 494 484

Product QuantiTect Primer Assay (200)


New

Contents For 200 x 50 l reactions; primer set in single tube* For 20 x 50 l reactions; primer sets in 96-well plate* For 100 x 50 l reactions; primer sets in 96-well plate* For 20 x 50 l reactions; primer sets in 384-well plate*

Cat. no. Varies Varies Varies Varies

QuantiTect Primer Assay 96 Plate (20) QuantiTect Primer Assay 96 Plate (100) QuantiTect Primer Assay 384 Plate (20)

New

New

* Includes a lyophilized mix of forward and reverse primers, which provides 10x QuantiTect Primer Assay when reconstituted.

Minimum order of 24 assays per plate. Minimum order of 96 assays per plate.

Further information and online ordering: www.qiagen.com/PG/QuantiTectPrimer

488

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

New

Rotor-Gene SYBR Green Kits

A
0.8 Normalized fluorescence
dF/dT

Amplification plot (QIAGEN)


1.2 1.0

For ultrafast, precise real-time PCR and RT-PCR using SYBR Green I

0.6 0.4 0.2 0.0 0

0.8 0.6 0.4 0.2 0.0 50 60 70 80 90 Temperature (C) 100

Ultrafast, reliable results on Rotor-Gene cyclers Specific detection of even low copy numbers Accurate detection of a wide range of template amounts Specially formulated master mix for fast cycling Guaranteed performance with QuantiTect Primer Assays
Product description Rotor-Gene SYBR Green Kits are dedicated for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly specific quantification in real-time PCR and RT-PCR using SYBR Green I. The novel PCR additive Q-Bond greatly reduces PCR run times (for more information, see page 492), while the cyclers unique rotary design delivers precise results through high thermal and optical uniformity from tube to tube. Highly specific amplification is achieved using a specially optimized master mix containing a balanced combination of K+ and NH4+ ions that promotes highly specific primer annealing (for more information, see page 492), and HotStarTaq Plus DNA Polymerase, which provides a fast and highly stringent hot start. The ready-to-use master mix also includes SYBR Green I dye and a dNTP mix (dATP, dCTP, dGTP, and dTTP), and can be conveniently stored at 28C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides fast and efficient cDNA synthesis. Two kit formats are available. For real-time PCR of genomic DNA or cDNA targets, choose the Rotor-Gene SYBR Green PCR Kit. For real-time one-step RT-PCR of RNA targets (i.e., reverse transcription followed by PCR in the same tube), use the Rotor-Gene SYBR Green RT-PCR Kit. Applications Rotor-Gene SYBR Green Kits are well suited for gene expression analysis using cDNA or RNA templates and for applications to quantify genomic DNA targets. The kits are compatible with the Rotor-Gene Q, Rotor-Gene 6000, and Rotor-Gene 3000.
B

10 15 20 25 30 35 40 45 Cycles

Amplification plot (Supplier R)

Specific and sensitive detection using SYBR Green. Tenfold dilutions of human leukocyte RNA (100 ng to 10 pg) were used as template in SYBR Green-based real-time one-step RT-PCR. Duplicate reactions were run using the QuantiTect Primer Assay for BCL2 (B-cell CLL/lymphoma 2). A The Rotor-Gene Q and Rotor-Gene SYBR Green RT-PCR Kit provided sensitive detection from 10 pg RNA and amplication of specic PCR product (melting curve shown in inset). B In contrast, an instrument and kit from Supplier R provided detection only after optimization of Mg2+ concentration. However, the limit of detection was 100 pg RNA and coamplication of nonspecic products was observed (melting curve shown in inset).

Product Guide 201011

Sample & Assay Technologies

489

PCR Assay Technologies

10.2

Superior performance in RT-PCR with SYBR Green


CT BAX (BCL2-associated X protein) BCL2 (apoptosis gene) MYC (proto-oncogene) -Actin (housekeeping gene) 24.84 26.96 28.42 20.24 QIAGEN Mean deviation 0.05 0.05 0.21 0.03 CT Supplier AII Mean deviation 0.46 0.29 0.72 0.12

29.57 32.83 35.26 24.39

Human leukocyte cDNA (1 ng) was used as template in SYBR Green-based real-time two-step RT-PCR. Triplicate reactions were run using QuantiTect Primer Assays for 4 different targets: BAX, BCL2, MYC, and -Actin. The Rotor-Gene Q and Rotor-Gene SYBR Green PCR Kit provided highly sensitive detection, indicated by lower CT values and lower mean deviations compared to an instrument and kit from Supplier AII.

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Reaction setup Real-time cycler DNA purification RNA purification cDNA synthesis QIAgility Rotor-Gene Q QIAamp DNA Blood Kits RNeasy Kits QuantiTect Reverse Transcription Kit QuantiTect Whole Transcriptome Kit FastLane Cell cDNA Kit QuantiTect Primer Assays 67 78 267 409 486 445 483 487

Predesigned primer sets

Product Rotor-Gene SYBR Green PCR Kit (80) Rotor-Gene SYBR Green PCR Kit (400) Rotor-Gene SYBR Green RT-PCR Kit (400)

Contents For 80 x 25 l reactions* For 400 x 25 l reactions* For 400 x 25 l reactions

Cat. no. 204072 204074 204174

* Includes 2x Rotor-Gene SYBR Green PCR Master Mix and RNase-free water. Includes 2x Rotor-Gene SYBR Green RT-PCR Master Mix, Rotor-Gene RT Mix, and RNase-free water.

Further information and online ordering: www.qiagen.com/PG/RotorGeneSYBRGreen

490

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

QuantiFast SYBR Green Kits


For fast real-time PCR and RT-PCR using SYBR Green I

Copies 1000 100 50 25 12 6 3 1 6

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

3 1

32 33 34 35 36

Resolution of small differences in copy number. The QuantiFast SYBR Green PCR Kit was used to detect the Y-chromosome-specic single-copy gene SRY in genomic DNA from a male donor on the Mastercycler ep realplex. Curves for 1000 copies down to 1 copy can be clearly distinguished from each other.

Product description QuantiFast SYBR Green Kits deliver fast and highly specific quantification in real-time PCR and RT-PCR using SYBR Green I. Fast cycling without compromising performance is achieved using the patent-pending PCR additive Q-Bond. Cycling times are reduced not only on fast cyclers with short ramping times, but also on standard cyclers. Highly specific amplification is achieved using a specially optimized master mix containing a balanced combination of K+ and NH4+ ions that promotes highly specific primer annealing, and HotStarTaq Plus DNA Polymerase, which provides a fast and highly stringent hot start. The ready-to-use master mix also includes SYBR Green I dye, a dNTP mix (dATP, dCTP, dGTP, and dTTP), and an optimized concentration of ROX dye for fluorescence normalization on certain cyclers (e.g., instruments from Applied Biosystems). The master mix can be conveniently stored at 28C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides fast and efficient cDNA synthesis.
Product Guide 201011 Sample & Assay Technologies 107 106 105 104 103 102 10

Detection over 7 logs of template in one-step RT-PCR. The QuantiFast SYBR Green RT-PCR Kit provided accurate quantication of HSP89 (a heat shock protein) transcript over a wide dynamic range. Reactions were run in triplicate on the Applied Biosystems 7500 Fast System using tenfold dilutions of in vitro transcript (107 down to 10 copies).

491

PCR Assay Technologies

10.2

Stabilization

Destabilization NH4+ NH3 + H+


K+

K+

Two kit formats are available. For real-time PCR of genomic DNA or cDNA targets, choose the QuantiFast SYBR Green PCR Kit. For real-time one-step RT-PCR of RNA targets (i.e., reverse transcription followed by PCR in the same tube), use the QuantiFast SYBR Green RT-PCR Kit. Principle Q-Bond increases the affinity of Taq DNA polymerases for short single-stranded DNA, reducing the time required for primer annealing to a few seconds. In addition, the unique composition of QuantiFast buffers supports the melting behavior of DNA, enabling short denaturation and annealing/extension times. The balanced combination of K+ and NH4+ ions promotes a high ratio of specific to nonspecific primer binding during the annealing step of each PCR cycle. This creates stringent primer annealing conditions, leading to increased PCR specificity. Primer annealing is only marginally influenced by the MgCl2 concentration, so optimization by titration of Mg2+ is not required. Procedure QuantiFast SYBR Green Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers and genomic DNA, cDNA, or RNA template to the ready-to-use master mix, and start the reaction on any real-time cycler.

Template

Primer

Specific primer annealing using a balanced combination of K+ and NH4+ ions. 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 2 cations maintains the high ratio of specic to nonspecic primertemplate binding over a wide temperature range.

Standard cycling
3' 5'

Fast primer annealing with Q-Bond. A Under standard-cycling conditions, denaturation, annealing, and extension usually occur as a 3-step process where the template is rst denatured, followed by primer annealing to form a binary complex and then polymerase binding to form a tertiary complex. B With Q-Bond in the reaction, this 3-step process becomes a faster 2-step process, where the template is rst denatured and the tertiary complex is then formed by the simultaneous binding of primer and polymerase, allowing extension to start within seconds.

5' 3'
B

Fast cycling
5' 5' 3' 3' Highly specific annealing

Q-Bond molecule

Taq DNA polymerase

Template DNA

Primer

492

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

Applications QuantiFast SYBR Green Kits are well suited for gene expression analysis using cDNA or RNA templates and for applications to quantify genomic DNA targets. The kits are compatible with any real-time cycler, including instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene. Alternative real-time PCR kits
Special feature Fast real-time PCR using probes Ultrafast real-time PCR using probes Fast multiplex real-time PCR using probes Ultrafast multiplex real-time PCR using probes Detection of microRNAs Sensitive detection of viruses Detection of SNPs and mutations Quantification of methylation status Description Master mix containing Q-Bond Master mix containing Q-Bond, for Rotor-Gene cyclers Master mix containing Q-Bond and Factor MP Master mix containing Q-Bond and Factor MP, for Rotor-Gene cyclers Complete system for miRNA/mRNA detection Highly concentrated 5x master mix Master mix developed for reliable and clear allelic discrimination Master mix for reliable quantification of methylation status Recommended kit Page 499 497 510 508 558 505 507 163

QuantiFast Probe Kits Rotor-Gene Probe Kits QuantiFast Multiplex Kits Rotor-Gene Multiplex Kits miScript PCR System QuantiTect Virus Kits Type-it Fast SNP Probe PCR Kit EpiTect MethyLight PCR Kits

Related products
Reaction setup DNA purification RNA purification cDNA synthesis QIAgility QIAamp DNA Blood Kits RNeasy Kits QuantiTect Reverse Transcription Kit QuantiTect Whole Transcriptome Kit FastLane Cell cDNA Kit QuantiTect Primer Assays 67 267 409 486 445 483 487

Predesigned primer sets

Product QuantiFast SYBR Green PCR Kit (80) QuantiFast SYBR Green PCR Kit (400) QuantiFast SYBR Green PCR Kit (2000) QuantiFast SYBR Green RT-PCR Kit (400) QuantiFast SYBR Green RT-PCR Kit (2000)

Contents For 80 x 25 l reactions* For 400 x 25 l reactions* For 2000 x 25 l reactions* For 400 x 25 l reactions For 2000 x 25 l reactions

Cat. no. 204052 204054 204056 204154

204156
Includes 2x QuantiFast

* Includes 2x QuantiFast SYBR Green PCR Master Mix and RNase-free water. SYBR Green RT-PCR Master Mix, QuantiFast RT Mix, and RNase-free water.

Master mix supplied in single tube.

Further information and online ordering: www.qiagen.com/PG/QuantiFastSYBRGreen

Product Guide 201011

Sample & Assay Technologies

493

PCR Assay Technologies

10.2

QuantiTect SYBR Green PCR Kit

QuantiTect SYBR Green Kits


For real-time PCR and RT-PCR using SYBR Green I


Supplier AII

High PCR specificity with integrated hot start No need to optimize reaction and cycling conditions Reliable quantification of low-abundance transcripts Accurate quantification over several logs of template Available with or without uracil-N-glycosylase (UNG)

Product description QuantiTect SYBR Green Kits provide highly specific detection in SYBR Green-based real-time PCR and RT-PCR on any real-time cycler. Reactions can be pretreated with uracil-N-glycosylase (UNG) to prevent potential carryover of PCR products from previous reactions. Highly specific amplification is achieved using an optimized master mix containing a balanced combination of K+ and NH4+ ions that promotes highly specific primer annealing (for more information, see page 492), and HotStarTaq DNA Polymerase, which provides a highly stringent hot start. The ready-to-use master mix also includes SYBR Green I dye, ROX passive reference dye, and a dNTP mix (dATP, dCTP, dGTP, and dTTP/ dUTP), and can be conveniently stored at 28C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides efficient cDNA synthesis. Three kit formats are available. For real-time PCR of genomic DNA or cDNA targets, choose either the QuantiTect SYBR Green PCR Kit (which does not include UNG) or the QuantiTect SYBR Green PCR +UNG Kit (which includes a UNG solution optimized for use with QuantiTect chemistries). For real-time one-step RT-PCR of RNA targets (i.e., reverse transcription followed by PCR in the same tube), use the QuantiTect SYBR Green RT-PCR Kit (optional UNG must be purchased separately).

Nonspecific products

No distinction between 1 pg template and NTC

Specific and sensitive quantification in two-step RT-PCR. Dilutions of human leukocyte cDNA (equivalent to 10 ng, 1 ng, 100 pg, 10 pg, and 1 pg RNA) were analyzed on the ABI PRISM 7000 using the QuantiTect Primer Assay for human IL8 and the indicated kits. Insets show melting curve analysis performed on the LightCycler 480. NTC: no template control.

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Product Guide 201011

Real-time PCR and RT-PCR

QuantiTect SYBR Green RT-PCR Kit (one-step RT-PCR)

10.2

10 ng (CT = 25.29) 1 ng (CT = 29.00) 100 pg (CT = 33.01)

Applications QuantiTect SYBR Green Kits are well suited for gene expression analysis using cDNA or RNA templates and for applications to quantify genomic DNA targets. The kits are compatible with any real-time cycler, including Rotor-Gene cyclers as well as instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene.
QuantiTect SYBR Green RT-PCR Kit (one-step RT-PCR) QuantiTect SYBR Green PCR Kit (two-step RT-PCR)

10 ng (CT = 25.29) 1 ng (CT = 29.00) 100 pg (CT = 33.01)

10 ng (CT = 25.49) 1 ng (CT = 29.32) 100 pg (CT = 32.91)

Comparable CT values in one-step and two-step RT-PCR. Total RNA (10 ng to 100 pg) or the equivalent amounts of cDNA from HeLa cells were analyzed QuantiTect SYBR Green PCR Kit (two-step RT-PCR) on the LightCycler 2.0 using the QuantiTect Primer Assay for human MAPK14 and the indicated kits.
10 ng (CT = 25.49) 1 ng (CT = 29.32) 32.91) 100 pg (CT =QIAGEN

Supplier R

Nonspecific products

High specificity in real-time PCR with UNG pretreatment. Real-time PCR with UNG pretreatment was carried out using either A the QuantiTect SYBR Green PCR +UNG Kit or B a kit and uracil-N-glycosylase from Supplier R. Reactions were run in duplicate on the LightCycler 480 using tenfold dilutions of human leukocyte cDNA (100 ng to 100 pg) and primers specic for Myc (a protooncogene). Melting curve analysis (see insets) revealed higher PCR specicity with the QuantiTect Kit than with the kit from Supplier R.

Product Guide 201011

Sample & Assay Technologies

495

PCR Assay Technologies

10.2

Related products
Reaction setup DNA purification RNA purification cDNA synthesis QIAgility QIAamp DNA Blood Kits RNeasy Kits QuantiTect Reverse Transcription Kit QuantiTect Whole Transcriptome Kit FastLane Cell cDNA Kit Predesigned primer sets QuantiTect Primer Assays 67 267 409 486 445 483 487

Product QuantiTect SYBR Green PCR Kit (40) QuantiTect SYBR Green PCR Kit (200) QuantiTect SYBR Green PCR Kit (1000) QuantiTect SYBR Green PCR +UNG Kit (200) QuantiTect SYBR Green RT-PCR Kit (200) QuantiTect SYBR Green RT-PCR Kit (1000)

Contents For 40 x 50 l reactions* For 200 x 50 l reactions* For 1000 x 50 l reactions* For 200 x 50 l reactions For 200 x 50 l reactions

Cat. no. 204141 204143 204145 204163 204243 204245

For 1000 x 50 l reactions

* Includes 2x QuantiTect SYBR Green PCR Master Mix and RNase-free water. Master mix supplied in single tube. Includes 2x QuantiTect SYBR Green PCR Master Mix, UNG, and RNase-free water. Includes 2x QuantiTect SYBR Green RT-PCR Master Mix, QuantiTect RT Mix, and RNase-free water.

Further information and online ordering: www.qiagen.com/PG/QuantiTectSYBRGreen

496

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

Normalized fluorescence

Normalized fluorescence

New

Rotor-Gene Probe Kits

0.6 0.5 0.4 0.3 0.2 0.1 0.0 5 15 25 Cycle 35 40

For ultrafast, precise real-time PCR and RT-PCR using sequence-specific probes

0.8 0.6 0.4 0.2 0.0

Normalized fluorescence

Normalized fluorescence

Ultrafast, reliable results on Rotor-Gene cyclers Sensitive detection of even low copy numbers Accurate detection of a wide range of template amounts Specially formulated master mix for fast 0.6 cycling
Product description
0.5 0.4 0.3 Rotor-Gene Probe Kits are dedicated for use with the 0.2 Rotor-Gene Q and other Rotor-Gene cyclers, providing 0.1 ultrafast, highly sensitive quantification 0.0 in real-time PCR and 15 25 35 RT-PCR using sequence-specific probes (e.g., 5 TaqMan probes). Cycle The novel PCR additive Q-Bond greatly reduces PCR run times (for more information, see page 492), while the cyclers unique rotary design delivers precise results through high thermal and optical uniformity from tube to tube.

0.8 0.6 0.4 0.2 0.0 10 20 Cycle 30 40

40

Highly sensitive and specific amplification is achieved using a specially optimized master mix containing a balanced combination of K+ and NH4+ ions that promotes highly specific primer annealing (for more information, see page 492), and HotStarTaq Plus DNA Polymerase, which provides a fast and highly stringent hot start. The ready-to-use master mix also includes a dNTP mix (dATP, dCTP, dGTP, and dTTP), and can be conveniently stored at 28C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides fast and efficient cDNA synthesis. Two kit formats are available. For real-time PCR of genomic DNA or cDNA targets, choose the Rotor-Gene Probe PCR Kit. For real-time one-step RT-PCR of RNA targets (i.e., reverse transcription followed by PCR in the same tube), use the Rotor-Gene Probe RT-PCR Kit. Applications Rotor-Gene Probe Kits are well suited for gene expression analysis using cDNA or RNA templates and for applications to quantify genomic DNA targets. The kits are compatible with the Rotor-Gene Q, Rotor-Gene 6000, and Rotor-Gene 3000.

Precise real-time PCR analysis. A Twofold dilutions of human genomic DNA from 30 ng (10,000 copies) to 0.06 ng (20 copies) were used as template in real-time PCR. Five replicate reactions were run for each dilution using a self-designed TaqMan assay for IL1R2 and the Rotor-Gene Probe PCR Kit on the Rotor-Gene Q. The average difference in the CT values between all dilutions was 1.07 cycles. B Human genomic DNA was used as template in 72 replicate real-time PCRs using a self-designed TaqMan assay for BCL2 on the Rotor-Gene Q without ROX normalization. The average CT value was 24.94 with a standard deviation of only 0.05, equivalent to a CV of 0.2%.

Product Guide 201011

Sample & Assay Technologies

497

PCR Assay Technologies

10.2

QIAGEN

B
6 E-1

Supplier AII

Normalized fluorescence

0.30

5 E-1

0.20

Rn
10 20 Cycle 30 40

4 E-1 3 E-1 2 E-1 1 E-1

0.10

0.00

0 0 5 10 15 20 Cycle 25 30 35 40

Sensitive detection using sequence-specific probe. Tenfold dilutions of human leukocyte RNA (100 ng to 1 ng) were used as template in real-time one-step RT-PCR. Triplicate reactions were run using a TaqMan Gene Expression Assay for IL12RB1 (interleukin 12 receptor, beta 1). Greater sensitivity (i.e., lower CT values) was achieved with A the Rotor-Gene Q and Rotor-Gene Probe RT-PCR Kit than with B an instrument and kit from Supplier AII.

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Virus detection

Product Rotor-Gene Probe PCR Kit (80) Rotor-Gene Probe PCR Kit (400) Rotor-Gene Probe RT-PCR Kit (400)

Contents For 80 x 25 l reactions* For 400 x 25 l reactions* For 400 x 25 l reactions

Cat. no. 204372 204374 204574

* Includes 2x Rotor-Gene Probe PCR Master Mix and RNase-free water. Includes 2x Rotor-Gene Probe RT-PCR Master Mix, Rotor-Gene RT Mix, and RNase-free water.

Further information and online ordering: www.qiagen.com/PG/RotorGeneProbe

498

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

QuantiFast Probe Kits


For fast real-time PCR and RT-PCR using sequence-specific probes

QuantiFast Probe PCR Kit (IL8 transcript)

Faster results with time savings of up to 60% One procedure for all standard and fast cyclers 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
Product description QuantiFast Probe Kits deliver fast and highly sensitive quantification in real-time PCR and RT-PCR using sequence-specific probes (e.g., TaqMan probes). Fast cycling without comprising performance is achieved using the patent-pending PCR additive Q-Bond (for more information, see page 492). Cycling times are reduced not only on fast cyclers with short ramping times, but also on standard cyclers. Highly sensitive and specific amplification is achieved using a specially optimized master mix containing a balanced combination of K+ and NH4+ ions that promotes highly specific primer annealing (for more information, see page 492), and HotStarTaq Plus DNA Polymerase, which provides a fast and highly stringent hot start. The ready-to-use master mix also includes a dNTP mix (dATP, dCTP, dGTP, and dTTP), and can be conveniently stored at 28C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides fast and efficient cDNA synthesis. Four kit formats are available. For cyclers that require a high ROX concentration for fluorescence normalization (i.e., all instruments from Applied Biosystems except the Applied Biosystems 7500), choose either the QuantiFast Probe PCR Kit (for real-time PCR of genomic DNA and cDNA targets) or the QuantiFast Probe RT-PCR Kit (for real-time one-step RT-PCR of RNA targets in a single tube). Both kits include a master mix containing ROX dye at an optimized concentration.
B

0.01 ng cDNA

Kit from Supplier I (IL8 transcript)

1 ng cDNA

Sensitive two-step RT-PCR. Reactions were run in triplicate on the ABI PRISM 7900 using tenfold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a TaqMan assay for IL8 (a chemokine). A The QuantiFast Kit showed greater sensitivity than B the kit from Supplier I (which was used according to the standard-cycling protocol), providing lower CT values and transcript quantication from as little as 0.01 ng cDNA.

Product Guide 201011

Sample & Assay Technologies

499

PCR Assay Technologies

10.2

For other cyclers, use either the QuantiFast Probe PCR +ROX Vial Kit (for real-time PCR of genomic DNA and cDNA targets) or the QuantiFast Probe RT-PCR +ROX Vial Kit (for real-time one-step RT-PCR of RNA targets in a single tube). Both kits contain ROX dye as a separate solution that is added to reactions if necessary. Applications QuantiFast Probe Kits are well suited for gene expression analysis using cDNA or RNA templates and for applications to quantify genomic DNA targets. The kits are compatible with any real-time cycler, including instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene.
A QuantiFast Probe RT-PCR +ROX Vial Kit (IL12RBI transcript) B Kit from Supplier I (IL12RBI transcript)

0.1 ng RNA

1 ng RNA

Sensitive one-step RT-PCR. Reactions were run in duplicate on the Applied Biosystems 7500 Fast System using tenfold dilutions of human leukocyte RNA (100 ng to 0.1 ng) and a TaqMan assay for IL12RBI (a type I transmembrane protein). The A QuantiFast Kit showed greater sensitivity than the B kit from Supplier I (which was used according to the standard-cycling protocol), providing lower CT values and transcript quantication from 0.1 ng RNA.

Two-step RT-PCR
Time savings L A1 A2 A3 0 20 40 60 80 100 120 46% 41% 58% 40%
QuantiFast QuantiTect

One-step RT-PCR
Time savings L A1 A2 A3 0 20 40 60 44% 44% 56% 41% 80 100 120 140
QuantiFast QuantiTect

Time (min)

Time (min)

Significantly reduced PCR times. QuantiFast Probe Kits reduce total PCR run time by up to 60% in A real-time two-step RT-PCR and B real-time one-step RT-PCR on standard cyclers (40 PCR cycles carried out; comparison with a standard QIAGEN real-time PCR kit). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000.

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

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Product QuantiFast Probe PCR Kit (80) QuantiFast Probe PCR Kit (400) QuantiFast Probe PCR Kit (2000) QuantiFast Probe PCR +ROX Vial Kit (80) QuantiFast Probe PCR +ROX Vial Kit (400) QuantiFast Probe PCR +ROX Vial Kit (2000) QuantiFast Probe RT-PCR Kit (400) QuantiFast Probe RT-PCR Kit (2000) QuantiFast Probe RT-PCR +ROX Vial Kit (400) QuantiFast Probe RT-PCR +ROX Vial Kit (2000)

Contents For 80 x 25 l reactions* For 400 x 25 l reactions* For 2000 x 25 l reactions*

Cat. no. 204252 204254 204256 204352 204354 204356 204454 204456 204554 204556

For 80 x 25 l reactions For 400 x 25 l reactions For 2000 x 25 l reactions

For 400 x 25 l reactions For 2000 x 25 l reactions For 400 x 25 l reactions

For 2000 x 25 l reactions

* For use with all instruments from Applied Biosystems except the Applied Biosystems 7500. Includes 2x QuantiFast Probe PCR Master Mix (with ROX dye) and RNase-free water.

Master mix supplied in single tube. For use with the Applied Biosystems 7500 and instruments from Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene. Includes 2x QuantiFast Probe PCR Master Mix (without ROX dye), 50x ROX Dye Solution, and RNase-free water. For use with all instruments from Applied Biosystems except the Applied Biosystems 7500. Includes 2x QuantiFast Probe RT-PCR Master Mix (with ROX dye), QuantiFast RT Mix, and RNase-free water. For use with the Applied Biosystems 7500 and instruments from Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene. Includes 2x QuantiFast Probe RT-PCR Master Mix (without ROX dye), 50x ROX Dye Solution, QuantiFast RT Mix, and RNase-free water.

Further information and online ordering: www.qiagen.com/PG/QuantiFastProbe Product Guide 201011 Sample & Assay Technologies 501

PCR Assay Technologies

10.2

QuantiTect Probe Kits


For real-time PCR and RT-PCR using sequence-specific probes

Highly sensitive detection of low-copy targets Accurate quantification over several logs of template No need to optimize reaction and cycling conditions Use of any sequence-specific probe on any real-time cycler Available with or without uracil-N-glycosylase (UNG)
Wide dynamic range in two-step RT-PCR. Duplicate reactions were performed on the Mx3005P using tenfold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a TaqMan assay for IL1R2 (a cytokine). The QuantiTect Probe PCR Kit provided accurate gene expression analysis from low to high template amounts with a PCR efciency of 101%.

Product description QuantiTect Probe Kits provide highly sensitive detection in probe-based real-time PCR and RT-PCR on any real-time cycler. Reactions can be pretreated with uracil-N-glycosylase (UNG) to prevent potential carryover of PCR products from previous reactions. The kits are designed for use with all types of sequence-specific probe, including TaqMan probes, FRET probes, and Molecular Beacons. Highly sensitive and specific amplification is achieved using an optimized master mix containing a balanced combination of K+ and NH4+ ions that promotes highly specific primer annealing (for more information, see page 492), and HotStarTaq DNA Polymerase, which provides a highly stringent hot start. The ready-to-use master mix also includes ROX passive reference dye and a dNTP mix (dATP, dCTP, dGTP, and dTTP/dUTP), and can be conveniently stored at 28C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides efficient cDNA synthesis. Three kit formats are available. For real-time PCR of genomic DNA or cDNA targets, choose either the QuantiTect Probe PCR Kit (which does not include UNG) or the QuantiTect Probe PCR +UNG Kit (which includes a UNG solution optimized for use with QuantiTect chemistries). For real-time one-step RT-PCR of RNA targets (i.e., reverse transcription followed by PCR in the same tube), use the QuantiTect Probe RT-PCR Kit (optional UNG must be purchased separately).

1 pg

Wide dynamic range in one-step RT-PCR. The QuantiTect Probe RT-PCR Kit provided accurate gene expression analysis from low to high template amounts. Duplicate reactions were performed on the ABI PRISM 7900 using tenfold dilutions of HeLa cell RNA (100 ng to 0.001 ng) and a TaqMan assay for 28S rRNA.

502

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

Applications QuantiTect Probe Kits are well suited for gene expression analysis using cDNA or RNA templates and for applications to quantify genomic DNA targets. The kits are compatible with any real-time cycler, including Rotor-Gene cyclers as well as instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene.
A A
QIAGEN QIAGEN

Supplier AII AII Supplier

PCR efficiency: 94% 94% PCR efficiency:

PCR efficiency: 83% 83% PCR efficiency:

CT = 34.64 CT = 34.64

CT = 36.92 CT = 36.92

Wide dynamic range in real-time PCR with UNG pretreatment. Real-time PCR with UNG pretreatment was carried out using either A the QuantiTect Probe PCR +UNG Kit or B a UNG-containing kit from Supplier AII. Reactions were run in duplicate on the ABI PRISM 7900 using tenfold dilutions of human leukocyte cDNA (100 ng to 10 pg) and a TaqMan assay for IL8 (a cytokine). The QuantiTect Kit provided lower CT values (i.e., greater sensitivity) and a higher PCR efciency than the kit from Supplier AII.

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Product Guide 201011

Sample & Assay Technologies

503

PCR Assay Technologies

10.2

Product QuantiTect Probe PCR Kit (40) QuantiTect Probe PCR Kit (200) QuantiTect Probe PCR Kit (1000) QuantiTect Probe PCR +UNG Kit (200) QuantiTect Probe RT-PCR Kit (200) QuantiTect Probe RT-PCR Kit (1000)

Contents For 40 x 50 l reactions* For 200 x 50 l reactions* For 1000 x 50 l reactions* For 200 x 50 l reactions

Cat. no. 204341 204343 204345 204363 204443 204445

For 200 x 50 l reactions For 1000 x 50 l reactions

* Includes 2x QuantiTect Probe PCR Master Mix and RNase-free water. Master mix supplied in single tube. Includes 2x QuantiTect Probe PCR Master Mix, UNG, and RNase-free water. Includes 2x QuantiTect Probe RT-PCR Master Mix, QuantiTect RT Mix, and RNase-free water.

Further information and online ordering: www.qiagen.com/PG/QuantiTectProbe 504 www.qiagen.com Product Guide 201011

Real-time PCR and RT-PCR

10.2

QuantiTect Virus Kits


For highly sensitive detection of viral RNA and/or DNA

ND 40 30 20 10 3 2.3 QuantiTect Virus Kit Supplier AII Supplier I 1.6 0.9 0.2 0.5 1.2 Template dilution Improved detection of low amounts of viral RNA compared with other real-time kits. Viral RNA was diluted in serial vefold dilutions and amplied in duplex with an internal control using the QuantiTect Virus Kit or kits from Suppliers AII and I. Reactions were run on the Applied Biosystems 7500. The QuantiTect Virus Kit provided much higher sensitivity than the other reagents tested, enabling reliable analysis of unknown samples. ND: Not detected after 50 PCR cycles. CT

High sensitivity in singleplex and multiplex assays Detection of viral RNA and/or DNA in the same reaction Clear detection of weak positive signals Fast universal 2-step protocols 5x master mix for higher sensitivity with more sample input
Product description The QuantiTect Virus Kit is specially designed for highly sensitive detection of viral nucleic acids. Multiplex assays enable detection of up to 4 viral RNA and/or DNA targets plus internal controls without loss of sensitivity. The supplied 5x master mix contains HotStarTaq Plus DNA Polymerase and dNTP mix in addition to ROX dye, at a concentration optimized for real-time cyclers that require a high ROX concentration in the amplification reaction. The supplied QuantiTect Virus RT Mix contains a unique formulation of Sensiscript Reverse Transcriptase optimized for highly sensitive detection of viral RNA. QuantiTect Nucleic Acid Dilution Buffer supplied with the kit stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips. The QuantiTect Virus +ROX Vial Kit is similar to the QuantiTect Virus Kit except that it includes a ROX-free 5x master mix and a separate vial of ROX dye. Applications QuantiTect Virus Kits provide highly sensitive real-time singleplex or multiplex PCR or one-step RT-PCR using sequence-specific probes for detection of viral DNA and/or RNA plus internal controls. The kits can be used on a wide range of real-time thermal cyclers.*
* Not intended for use with capillary thermal cyclers, such as LightCycler 1.x and LightCycler 2.0 instruments.

Product Guide 201011

Sample & Assay Technologies

505

PCR Assay Technologies

10.2

A
12000 10000 8000 6000 4000 2000 0 -2000

B
8000 7000 6000 5000 4000 3000 2000 1000 0 -1000

Unambiguous determination of CT values over a broad dynamic range. Serial dilutions of bovine viral diarrhea virus (BVDV) RNA, as indicated, were amplied and analyzed using the QuantiTect Virus Kit on the Applied Biosystems 7500. The steep sigmoidal curves enable accurate CT value determination even at low template amounts (1 copy/l). A Detection of BVDV1 RNA. B Detection of BVDV2 RNA. NTC: No template control.

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Product QuantiTect Virus Kit (50) QuantiTect Virus Kit (200) QuantiTect Virus Kit (1000) QuantiTect Virus +ROX Vial Kit (50) QuantiTect Virus +ROX Vial Kit (200) QuantiTect Virus +ROX Vial Kit (1000)

Contents For 50 x 50 l reactions* For 200 x 50 l reactions* For 1000 x 50 l reactions* For 50 x 50 l reactions

Cat. no. 211011 211013 211015 211031 211033 211035

For 200 x 50 l reactions For 1000 x 50 l reactions

* Includes QuantiTect Virus Master Mix (with ROX dye), QuantiTect Virus RT Mix, RNase-free water, and QuantiTect Nucleic Acid Dilution Buffer.

Includes QuantiTect Virus NR Master Mix (without ROX dye), ROX dye solution, QuantiTect Virus RT Mix, RNase-free water, and QuantiTect Nucleic Acid Dilution Buffer.

Further information and online ordering: www.qiagen.com/PG/QuantiTectVirus

506

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

Type-it Fast SNP Probe PCR Kit


For accurate and reliable SNP genotyping using TaqMan or TaqMan MGB probes

Validated using TaqMan SNP Genotyping Assays* Automated allele calling and tight fluorescence clusters Suitable for difficult SNP loci or very low template amounts Up to 40% time savings due to fast cycling procedure
Product description The Type-it Fast SNP Probe PCR Kit is based on highly specific HotStarTaq Plus DNA Polymerase and a newly developed buffer system, both of which enable reliable and clear allelic discrimination. The combination of all components provided in the master mix result in improved accuracy and highly specific probe binding. The Type-it Fast SNP Probe PCR Kit is validated using TaqMan SNP Genotyping Assays* and enables reproducible SNP genotyping even with difficult SNP loci (e.g., GC rich) or low amounts of starting template. Applications The Type-it Fast SNP Probe PCR Kit is dedicated for SNP genotyping using commercially available SNP genotyping assays based on TaqMan MGB probes or user-developed 5' nuclease probes. The kit is compatible with standard as well as fast ramping cyclers and real-time cyclers.
* Commercially available from other suppliers.

Outstanding separation and tight allele clustering. Cluster plot analysis using a TaqMan MGB-based SNP genotyping assay and a panel of 80 different genomic DNAs.

Product Type-it Fast SNP Probe PCR Kit (100) Type-it Fast SNP Probe PCR Kit (800) Type-it Fast SNP Probe PCR Kit (4000)

Contents For 100 x 25 l reactions For 800 x 25 l reactions For 4000 x 25 l reactions

Cat. no. 206042 206045 206047

Includes Type-it Fast SNP Probe PCR Master Mix (with optimized MgCl2 concentration and 200 M each dNTP), containing HotStarTaq Plus DNA Polymerase, 5x Q-Solution, and RNase-free water. Master mix supplied in 2 individual tubes.

Further information and online ordering: www.qiagen.com/PG/TypeitFastSNP Product Guide 201011 Sample & Assay Technologies 507

PCR Assay Technologies

10.2

IL8 (FAM)

New

Rotor-Gene Multiplex Kits

For ultrafast, precise multiplex real-time PCR and RT-PCR using sequence-specific probes

Ultrafast, reliable results on Rotor-Gene cyclers Sensitive detection of multiple targets in 1 tube Successful multiplex PCR without the need for optimization Precise discrimination of small differences in target amount

Product description
ACTB (HEX)

Rotor-Gene Multiplex Kits are dedicated for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly reliable quantification in multiplex, real-time PCR and RT-PCR using sequence-specific probes (e.g., TaqMan probes). The novel PCR additive Q-Bond greatly reduces PCR run times (for more information, see page 492), while the cyclers unique rotary design delivers precise results through high thermal and optical uniformity from tube to tube. Up to 4 targets (e.g., 1 control gene and 3 genes of interest) can be quantified simultaneously in the same reaction. Reliable multiplex analysis without the need for optimization is achieved using a specially optimized master mix. The master mix contains Factor MP and a balanced combination of K+ and NH4+ ions, which promote stable and specific primer annealing (for more information, see page 492), and HotStarTaq Plus DNA Polymerase, which provides a fast and highly stringent hot start. The ready-to-use master mix also includes a dNTP mix (dATP, dCTP, dGTP, and dTTP), and can be conveniently stored at 28C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides fast and efficient cDNA synthesis. Two kit formats are available. For real-time PCR of genomic DNA or cDNA targets, choose the Rotor-Gene Multiplex PCR Kit. For real-time one-step RT-PCR of RNA targets (i.e., reverse transcription followed by PCR in the same tube), use the Rotor-Gene Multiplex RT-PCR Kit.

CT values
CT values (IL8) CT values (ACTB) Duplex 14.78 18.20 21.77 25.50 29.01 32.88 45.00 Singleplex 14.25 17.23 21.26 24.98 28.48 32.36 45.00

Template 100 ng 10 ng 1 ng 100 pg 10 pg 1 pg NTC

Duplex 15.17 18.57 21.51 25.42 28.96 33.76 45.00

Singleplex 15.70 19.14 22.65 26.18 29.41 34.75 45.00

Reliable duplex analysis. Duplex, real-time two-step RT-PCR was carried on the Rotor-Gene Q using the Rotor-Gene Multiplex PCR Kit and self-designed TaqMan assays for A IL8 (interleukin 8) and B ACTB (-actin). Analysis of tenfold dilutions of leukocyte cDNA template from 100 ng to 1 pg provided high PCR efciencies of around 95%. C The CT values were comparable with those achieved in control singleplex reactions, demonstrating the reliability of the duplex assay.

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Product Guide 201011

Real-time PCR and RT-PCR

10.2

Applications Rotor-Gene Multiplex Kits are well suited for gene expression analysis using cDNA or RNA templates and for applications to quantify genomic DNA targets. The kits are compatible with the Rotor-Gene Q, Rotor-Gene 6000, and Rotor-Gene 3000.
GAPDH (HEX) RPS27A (CAL Fluor Red 610) 18S rRNA (Quasar 670) NFKB (Quasar 705)

35 30 25 20 15 10 5 0 0 1 2 3 4

log pg template

Highly efficient 4-plex analysis. 4-plex, real-time one-step RT-PCR was performed using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays for the indicated targets. Reactions were run on the Rotor-Gene Q using 100, 10, 1, or 0.1 ng RNA from HeLa cells. The plots of CT value versus log template amount were parallel, indicating all 4 targets were amplied with the same high efciency. GAPDH: glyceraldehyde-3phosphate dehydrogenase; RPS27A: ribosomal protein S27a; NFKB: nuclear factor of kappa light polypeptide gene enhancer in B-cells.

CT

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Product Rotor-Gene Multiplex PCR Kit (80) Rotor-Gene Multiplex PCR Kit (400) Rotor-Gene Multiplex RT-PCR Kit (80) Rotor-Gene Multiplex RT-PCR Kit (400)

Contents For 80 x 25 l reactions* For 400 x 25 l reactions* For 80 x 25 l reactions

Cat. no. 204772 204774 204972 204974

For 400 x 25 l reactions

* Includes 2x Rotor-Gene Multiplex PCR Master Mix and RNase-free water. Includes 2x Rotor-Gene Multiplex RT-PCR Master Mix, Rotor-Gene RT Mix, and RNase-free water.

Further information and online ordering: www.qiagen.com/PG/RotorGeneMultiplex Product Guide 201011 Sample & Assay Technologies 509

PCR Assay Technologies

10.2

QuantiFast Multiplex PCR +R Kit (fast-cycling mode)

QuantiFast Multiplex Kits


For fast multiplex real-time PCR and RT-PCR using sequencespecific probes

108

10

Sensitive detection of multiple targets in 1 well Faster results with time savings of up to 50% Successful multiplex PCR without the need for optimization Universal protocol for all standard and fast cyclers Precise discrimination of small differences in target amount

Kit from Supplier AII (standard-cycling mode)

Sensitive duplex PCR and wide dynamic range. Duplicate reactions were run on the Applied Biosystems 7500 Fast System using a DNA template mix providing 108 copies of -actin (data shown in insets) and 106 to 10 copies of RPS27A (a ribosomal protein). The A QuantiFast Multiplex PCR +R Kit showed clearly higher sensitivity than the B duplex PCR kit from Supplier AII, enabling the cycler in fast-cycling mode to detect 10 copies of target and quantify over 6 log dilutions of template.

510

Product description QuantiFast Multiplex Kits deliver fast and highly reliable quantification in multiplex, real-time PCR and RT-PCR using sequence-specific probes (e.g., TaqMan probes). Fast cycling without comprising performance is achieved using the patent-pending PCR additive Q-Bond (for more information, see page 492). Cycling times are reduced not only on fast cyclers with short ramping times, but also on standard cyclers. Up to 4 targets (e.g., 1 control gene and 3 genes of interest) can be quantified simultaneously in the same reaction. Reliable multiplex analysis without the need for optimization is achieved using a specially optimized master mix. The master mix contains Factor MP and a balanced combination of K+ and NH4+ ions, which promote stable and specific primer annealing (for more information, see page 492), and HotStarTaq Plus DNA Polymerase, which provides a fast and highly stringent hot start. The ready-to-use master mix also includes a dNTP mix (dATP, dCTP, dGTP, and dTTP), and can be conveniently stored at 28C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides fast and efficient cDNA synthesis. Four kit formats are available. For cyclers that require a high ROX concentration for fluorescence normalization (i.e., all instruments from Applied Biosystems except the Applied Biosystems 7500), choose either the QuantiFast Multiplex PCR Kit (for real-time PCR of genomic DNA and cDNA targets) or the QuantiFast Multiplex RT-PCR Kit (for real-time one-step RT-PCR of RNA targets in a single tube). Both kits include a master mix containing ROX dye at an optimized concentration.

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Real-time PCR and RT-PCR

10.2

For other cyclers, use either the QuantiFast Multiplex PCR +R Kit (for real-time PCR of genomic DNA and cDNA targets) or the QuantiFast Multiplex RT-PCR +R Kit (for real-time one-step RT-PCR of RNA targets in a single tube). Both kits contain ROX dye as a separate solution that is added to reactions if necessary. Applications QuantiFast Multiplex Kits are well suited for gene expression analysis using cDNA or RNA templates and for applications to quantify genomic DNA targets. The kits are compatible with any real-time cycler, including instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene.
A
RPS27A (Cy5 dye)

GAPDH (HEX dye)

UBC (FAM dye)

Comparable results in triplex and singleplex RT-PCR on the LightCycler 480. Triplex and singleplex, real-time one-step RT-PCR were carried out using the QuantiFast Multiplex RT-PCR +R Kit and self-designed TaqMan assays for A RPS27A (a ribosomal protein), B GAPDH (a housekeeping gene), and C UBC (a housekeeping gene). The template was Ramos cell line RNA (10 ng, 1 ng, or 0.1 ng), and reactions were run in duplicate. The comparable CT values for triplex PCR (colored curves) and singleplex PCR (gray curves) and the high PCR efciencies (data not shown) demonstrate the reliability of triplex PCR with the QuantiFast Multiplex RT-PCR +R Kit when analyzing targets of differing abundance.

Product Guide 201011

Sample & Assay Technologies

511

PCR Assay Technologies

10.2

Significantly reduced PCR times. QuantiFast Multiplex Kits reduce total PCR run time by up to 50% in A real-time two-step RT-PCR and B real-time one-step RT-PCR (40 cycles run; comparison with QuantiTect Multiplex Kits). I: iCycler iQ; L1: LightCycler 480; L2: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500 Fast System; M: Mx3005P.

Two-step RT-PCR
Time savings

One-step RT-PCR

I L1 L2 A1 A2 M 0 40 80 120 Time (min)

42% 42% 48% 41% 44% 39% 160 QuantiFast QuantiTect I L1 L2 A2 M 0 50 100 150 Time (min)

Time savings 38% 45% 48% 46% 41% 200 QuantiFast QuantiTect

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Virus detection

Product QuantiFast Multiplex PCR Kit (80) QuantiFast Multiplex PCR Kit (400) QuantiFast Multiplex PCR +R Kit (80) QuantiFast Multiplex PCR +R Kit (400)
New New New New

Contents For 80 x 25 l reactions* For 400 x 25 l reactions* For 80 x 25 l reactions For 400 x 25 l reactions For 80 x 25 l reactions

Cat. no. 204652 204654 204752 204754 204852 204854 204952

QuantiFast Multiplex RT-PCR Kit (80) QuantiFast Multiplex RT-PCR Kit (400) QuantiFast Multiplex RT-PCR +R Kit (80) QuantiFast Multiplex RT-PCR +R Kit (400)

For 400 x 25 l reactions For 80 x 25 l reactions

For 400 x 25 l reactions

204954

* For use with all instruments from Applied Biosystems except the Applied Biosystems 7500. Includes 2x QuantiFast Multiplex PCR Master Mix (with ROX dye) and RNase-free water.

For use with the Applied Biosystems 7500 and instruments from Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene. Includes 2x QuantiFast Multiplex PCR Master Mix (without ROX dye), 50x ROX Dye Solution, and RNase-free water. For use with all instruments from Applied Biosystems except the Applied Biosystems 7500. Includes 2x QuantiFast Multiplex RT-PCR Master Mix (with ROX dye), QuantiFast RT Mix, and RNase-free water. For use with the Applied Biosystems 7500 and instruments from Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene. Includes 2x QuantiFast Multiplex RT-PCR Master Mix (without ROX dye), 50x ROX Dye Solution, QuantiFast RT Mix, and RNase-free water.

Further information and online ordering: www.qiagen.com/PG/QuantiFastMultiplex

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Real-time PCR and RT-PCR

10.2

QuantiTect Multiplex Kits


For multiplex, real-time PCR and RT-PCR using sequencespecific probes

Target 1 (FAM)
Triplex PCR Singleplex PCR

Multiplex analysis with no need for optimization Analysis of multiple targets in a single reaction Sensitive detection of as few as 10 copies of each target Reliable quantification of target and reference genes Ready-to-use master mix compatible with all cyclers
Product description QuantiTect Multiplex Kits provide highly reliable multiplex real-time PCR and RT-PCR on any real-time cycler. Up to 5 targets (e.g., 1 control gene and 4 genes of interest) can be quantified simultaneously in the same reaction. Reactions can be pretreated with uracil-N-glycosylase (UNG) to prevent potential carryover of PCR products from previous reactions (UNG must be purchased separately). The kits are designed for use with all types of sequence-specific probe, including TaqMan probes and FRET probes. Reliable multiplex analysis without the need for optimization is achieved using a specially optimized master mix. The master mix contains Factor MP and a balanced combination of K+ and NH4+ ions, which promote stable and specific primer annealing (for more information, see page 492), and HotStarTaq DNA Polymerase, which provides a highly stringent hot start. The ready-to-use master mix also includes a dNTP mix (dATP, dCTP, dGTP, and dTTP/dUTP), and can be conveniently stored at 28C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides efficient cDNA synthesis. Four kit formats are available. For cyclers that require ROX passive reference dye for fluorescence normalization, choose either the QuantiTect Multiplex PCR Kit (for real-time PCR of genomic DNA and cDNA targets) or the QuantiTect Multiplex RT-PCR Kit (for real-time one-step RT-PCR of RNA targets in a single tube). Both kits include a master mix containing ROX dye.

Target 2 (HEX)
Triplex PCR Singleplex PCR

Target 3 (Cy5)
Triplex PCR Singleplex PCR

Equivalent CT values in triplex PCR and singleplex PCR. Dilutions of cDNA template (100 ng, 11.11 ng, 1.23 ng, and 0.14 ng) were analyzed by triplex PCR (colored curves) and by singleplex PCR (gray curves) on the iCycler iQ. TaqMan probes labeled with FAM, HEX, or Cy5 dye were used. (Data kindly provided by the University of Minnesota, Minneapolis, MN, USA.)

Product Guide 201011

Sample & Assay Technologies

513

PCR Assay Technologies

10.2

Variable amounts of target RNA (FAM)

1000 100 10

For other cyclers, use either the QuantiTect Multiplex PCR NoROX Kit (for real-time PCR of genomic DNA and cDNA targets) or the QuantiTect Multiplex RT-PCR NR Kit (for real-time one-step RT-PCR of RNA targets in a single tube). Both kits are supplied with a master mix free of ROX dye. Applications QuantiTect Multiplex Kits are well suited for gene expression analysis using cDNA or RNA templates and for applications to quantify genomic DNA targets. The kits are compatible with any real-time cycler, including Rotor-Gene cyclers as well as instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene.

Constant amounts of internal control (HEX)

1000
28S rRNA (triplex PCR) POLD3 (triplex PCR) CDK2 (triplex PCR) 28S rRNA, POLD3, or CDK2 (singleplex PCR)

Detection of down to 10 copies of target RNA in duplex PCR. Duplex, real-time one-step RT-PCR was performed on the Applied Biosystems 7500 using the QuantiTect Multiplex RT-PCR Kit and TaqMan probes. The target was A 1000, 100, or 10 copies of an in vitro transcript of a viral gene, each spiked with B 1000 copies of synthetic internal control RNA. Reactions were performed in replicate. Target RNA was detected using a FAM labeled probe. Internal control was detected using a HEX labeled probe. Seven replicates of 10 copies of target RNA were reproducibly detected.

Comparable results between triplex PCR and corresponding singleplex PCRs. Triplex, real-time one-step RT-PCR was performed on the Applied Biosystems 7500 using the QuantiTect Multiplex RT-PCR Kit and TaqMan probes (labeled with FAM, HEX, or Cy5 dye). The template was 20 ng total RNA from the Ramos cell line, and the targets were 28S rRNA, POLD3, and CDK2. Reactions were performed in triplicate. The resulting CT values are equivalent to those obtained when the targets were amplied and detected individually (black curves).

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Real-time PCR and RT-PCR

10.2

Related products
Reaction setup DNA purification RNA purification cDNA synthesis Virus detection QIAgility QIAamp DNA Blood Kits RNeasy Kits QuantiTect Reverse Transcription Kit QuantiTect Whole Transcriptome Kit FastLane Cell cDNA Kit QuantiTect Virus Kits 67 267 409 486 445 483 505

Product QuantiTect Multiplex PCR Kit (40) QuantiTect Multiplex PCR Kit (200) QuantiTect Multiplex PCR Kit (1000) QuantiTect Multiplex PCR NoROX Kit (40) QuantiTect Multiplex PCR NoROX Kit (200) QuantiTect Multiplex PCR NoROX Kit (1000) QuantiTect Multiplex RT-PCR Kit (200) QuantiTect Multiplex RT-PCR Kit (1000) QuantiTect Multiplex RT-PCR NR Kit (200) QuantiTect Multiplex RT-PCR NR Kit (1000)

Contents For 40 x 50 l reactions* For 200 x 50 l reactions* For 1000 x 50 l reactions*

Cat. no. 204541 204543 204545 204741 204743 204745 204643 204645 204843 204845

For 40 x 50 l reactions For 200 x 50 l reactions For 1000 x 50 l reactions

For 200 x 50 l reactions For 1000 x 50 l reactions For 200 x 50 l reactions

For 1000 x 50 l reactions

* For use with all instruments from Applied Biosystems. Includes 2x QuantiTect Multiplex PCR Master Mix (with ROX dye) and RNase-free water.

Master mix supplied in single tube. For use with Rotor-Gene cyclers and instruments from Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene. Includes 2x QuantiTect Multiplex PCR NoROX Master Mix (without ROX dye) and RNase-free water. For use with all instruments from Applied Biosystems. Includes 2x QuantiTect Multiplex RT-PCR Master Mix (with ROX dye), QuantiTect RT Mix, and RNase-free water. For use with Rotor-Gene cyclers and instruments from Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene. Includes 2x QuantiTect Multiplex RT-PCR NoROX Master Mix (without ROX dye), QuantiTect RT Mix, and RNase-free water.

Further information and online ordering: www.qiagen.com/PG/QuantiTectMultiplex Product Guide 201011 Sample & Assay Technologies 515

PCR Assay Technologies

10.2

New

Type-it HRM PCR Kit

For fast and accurate detection of gene mutations and SNPs by HRM analysis

Accurate detection of subtle sequence variations Highly specific amplification of difficult mutation loci Distinct melting curves due to EvaGreen fluorescent dye Fast and easy development of new HRM genotyping assays Convenient master mix format and optimized protocol
Product description The Type-it HRM PCR Kit is designed for fast and accurate genotyping using high-resolution melting (HRM) technology. The kit is available in a convenient master mix format that contains the innovative fluorescent dye EvaGreen, as well as a novel HRM buffer, HotStarTaq Plus DNA Polymerase, and dNTPs. The Type-it HRM PCR Kit is specially optimized to enable successful analysis of genomic loci that are difficult to amplify. Development of new HRM genotyping assays is straightforward due to elimination of time-consuming optimization of PCR parameters. Application The Type-it HRM PCR Kit provides a highly reliable and cost-effective method for various genotyping applications in several fields of research, such as disease association studies or cancer research. The kit is compatible with all real-time instruments suitable for HRM analysis. The Type-it HRM 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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Real-time PCR and RT-PCR

10.2

A
5 0 -5 -10 -15

2x HRM Master Mix (Q); 25 l/reaction

B
5 0 -5 -10 -15

2x HRM Master (Supplier R); 20 l/reaction

.5

.5

.0

.5

.0

.5

.0

.5

.0

.0

77

Clear separation from wild type

Successful scanning for gene mutations. The human KRAS gene AA 12 and 13 was scanned for mutations using the Type-it HRM PCR Kit. A Difference plot showing reliable discrimination between wild type sequence (blue) and the c.35 G>C mutation (brown curve), resulting in p.G12A and c.37G>T mutation (pink curve), resulting in p.G13C. Reliable discrimination with high condence was obtained without the need for optimization (see table for genotyping results). B The c.35 G>C mutation could not be resolved from the wild type even after extensive optimization of Mg2+ concentration and cycling parameters when using the HRM master mix from Supplier R.

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Product Type-it HRM PCR Kit (100)* Type-it HRM PCR Kit (400)*

.5 78 .0 78 .5 79 .0 79 .5 80 .0 80 .5 81 .0 81 .5 82 .0 82 .5
Incorrect genotyping

80

76

77

77

78

78

79

79

80

81

Contents For 100 reactions For 400 reactions

Cat. no. 206542 206544

* Includes 2x HRM Master Mix (contains HotStarTaq Plus DNA Polymerase, Type-it HRM PCR Buffer [with EvaGreen dye], optimized concentration of Q-Solution, MgCl2, and dNTP Mix) and RNase-free water.

Master mix supplied in 2 individual tubes.

Further information and online ordering: www.qiagen.com/PG/TypeitHRM Product Guide 201011 Sample & Assay Technologies 517

PCR Assay Technologies

10.3

120 100 80 60 40 20 0 0.5 kb 1 kb 3 kb

QIAGEN PCR Cloningplus Kit


For direct cloning of PCR products

Just 40 minutes from PCR product to plated cells Ready-to-use Ligation Master Mix Immediate plating of transformed competent cells High-specificity UA hybridization for efficient cloning Competent cells supplied with the kit
Product description The QIAGEN PCR Cloningplus Kit provides ready-to-use ligation reactions, which contain linearized cloning vectors that carry U overhangs at each 3' end, allowing PCR products containing 3'-end A overhangs to be directly ligated and cloned with high efficiency. The QIAGEN PCR Cloningplus Kit provides competent E. coli cells and SOC medium for efficient transformation. Applications The QIAGEN PCR Cloningplus Kit is suitable for cloning of any PCR product that has a single A overhang at each 3' end. PCR products generated using Taq DNA polymerase, other non-proofreading DNA polymerases, or the HotStar HiFidelity Polymerase Kit (page 468) can be directly cloned without any preparation. QIAGEN also offers the QIAexpress UA Cloning Kit (page 177), for direct cloning of PCR products into the pQE-30 UA vector for high-level expression of 6xHis-tagged proteins.

Highly specific cloning with a short 30-minute ligation time. The effect of ligation time on cloning efciency was compared for the QIAGEN PCR Cloningplus Kit and a TA-based cloning kit (Supplier I) using PCR products of different length (0.5 kb, 1 kb, and 3 kb). Colony numbers were converted to relative percentages, with the QIAGEN PCR Cloningplus Kit procedure set at 100% for each comparison. 30 min ligation (QIAGEN PCR Cloningplus Kit recommendation).

Product QIAGEN PCR Cloning QIAGEN PCR Cloning


plus plus

Contents Kit (10) Kit (40) For 10 reactions* For 40 reactions*

Cat. no. 231222 231224

* Includes 2x ligation master mix, pDrive cloning vector, distilled water, QIAGEN EZ competent cells, and SOC medium. Further information and online ordering: www.qiagen.com/PG/PCRCloningPlus 518 www.qiagen.com Product Guide 201011

Cloning

10.3

QIAGEN PCR Cloning Kit


For direct cloning of PCR products generated by Taq DNA polymerases

120 100 80 60

Fast and simple procedure Ready-to-use Ligation Master Mix High-specificity UA hybridization for efficient cloning
Product description The QIAGEN PCR Cloning Kit provides ready-to-use ligation reactions, which contain linearized cloning vectors that carry U overhangs at each 3' end, allowing PCR products containing 3'-end A overhangs to be directly ligated and cloned with high efficiency. Applications The QIAGEN PCR Cloning Kit is suitable for cloning of any PCR product that has a single A overhang at each 3' end. PCR products generated using Taq DNA polymerase, other non-proofreading DNA polymerases, or the HotStar HiFidelity Polymerase Kit (page 468) can be directly cloned without any preparation. QIAGEN also offers the QIAexpress UA Cloning Kit (page 177), for direct cloning of PCR products into the pQE-30 UA expression vector for high-level expression of 6xHis-tagged proteins.

40 20 0
ls F Q C IA om G pe EN te EZ nt C el 10 JM P 10 9

Robust QIAGEN EZ Competent Cells do not require recovery incubation. QIAGEN EZ Competent Cells (>108 cfu/g DNA), TOP 10F (Supplier I; >109 cfu/g DNA), and JM109 (Supplier P; >108 cfu/g DNA) competent cells were transformed with pUC18 plasmid DNA. The recommended protocol from each supplier was followed, except that all cells were plated immediately onto agar/ampicillin plates without a recovery incubation in SOC medium. Colony numbers were converted to relative percentages, with QIAGEN EZ Competent Cells set at 100%. Colony numbers were not normalized for transformation efciency. Normalization would result in an even higher transformation efciency for QIAGEN EZ Competent Cells. (QIAGEN EZ Competent Cells are supplied with the QIAGEN PCR Cloningplus Kit.)

Product QIAGEN PCR Cloning Kit (10) QIAGEN PCR Cloning Kit (40)

Contents For 10 reactions* For 40 reactions*

TO

Cat. no. 231122 231124

* Includes 2x ligation master mix, pDrive cloning vector, and distilled water. Further information and online ordering: www.qiagen.com/PG/PCRCloning

Product Guide 201011

Sample & Assay Technologies

519

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