Pcr

446 www.qiagen.com Product Guide 2010–11

From Sample to Result

PCR Assay Technologies1010.1 End-point PCR and RT-PCR

10.2 Real-time PCR and RT-PCR

10.3 Cloning

Product Guide 2010-11 - Chapter 10 - RoW Version - Page 446

Product Guide 2010–11 Sample & Assay Technologies 447

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



10.1 End-point PCR and RT-PCR www.qiagen.com/PG/PCR

■ Selection guides

■ End-point PCR: applications 452

■ End-point PCR: features 453

■ End-point RT-PCR: applications 454

■ End-point RT-PCR: features 454

■ PCR enzymes

■ Standard PCR, fridge storage TopTaq DNA Polymerase 455

■ Standard PCR Taq DNA Polymerase 457

■ Standard PCR, with dNTPs Taq PCR Core Kit 457

■ Hot-start PCR HotStarTaq Plus DNA Polymerase 460

HotStarTaq DNA Polymerase 462

■ Ready-to-use PCR master mixes

■ Standard PCR, fridge storage TopTaq Master Mix Kit 456

■ Standard PCR Taq PCR Master Mix Kit 459

■ Hot-start PCR HotStarTaq Plus Master Mix Kit 461

HotStarTaq Master Mix Kit 465

■ Rapid hot-start PCR QIAGEN Fast Cycling PCR Kit 466

■ dNTPs

■ PCR grade dNTP Set, PCR Grade 467

dNTP Mix, PCR Grade 467

■ PCR kits for challenging applications

■ High-fidelity, hot-start PCR HotStar HiFidelity Polymerase Kit 468

■ Long-range PCR QIAGEN LongRange PCR Kit 469

■ Multiplex, hot-start PCR QIAGEN Multiplex PCR Kit 470

■ Template amplification for Pyrosequencing analysis New PyroMark PCR Kit 471

■ Methylation-specific PCR (MSP) EpiTect MSP Kit 162

■ Methylation analysis using HRM analysis New EpiTect HRM PCR Kit 165



n PCR kits dedicated for genotyping applications

n Analysis of microsatellite, STR, and VNTR loci using multiplex PCR Type-it Microsatellite PCR Kit 472

n Detection of mutations using multiplex PCR Type-it Mutation Detect PCR Kit 473

n SNP genotyping using probe-based real-time PCR with fast cycling Type-it Fast SNP Probe PCR Kit 507

n Detection of gene mutations and SNPs using HRM analysis New Type-it HRM PCR Kit 516

n RT-PCR kits

n Sensitive one-step RT-PCR in one tube QIAGEN OneStep RT-PCR Kit 474n Accurate and long-range two-step

RT-PCR QIAGEN LongRange 2Step RT-PCR Kit 475

n Reverse-transcription enzymes

n Using 50 ng to 2 μg RNA Omniscript RT Kit 477

n Using less than 50 ng RNA Sensiscript RT Kit 478

n Automated solutions

n Reaction setup in all formats New QIAgility 67n Multicapillary electrophoresis of

PCR products QIAxcel System 83

n HRM analysis New Rotor-Gene Q 78

n DNA cleanup from PCR See PCR cleanup 362

n Whole genome amplification See Whole genome amplification 256

10.2 Real-time PCR and RT-PCR www.qiagen.com/PG/realtime

n Selection guides

n Real-time PCR and RT-PCR: ultrafast, precise quantification on Rotor-Gene cyclers 479

n Real-time PCR and RT-PCR: fast cycling 480

n Real-time PCR and RT-PCR: standard cycling 481

n Real-time RT-PCR: direct from cell lysates without RNA purification 482


für Seitenzahl

in DATAform



■ Real-time RT-PCR directly from cultured cells

■ From cells directly to cDNA FastLane Cell cDNA Kit 483

■ From cells directly to RT-PCR results FastLane Cell RT-PCR Kits 484

■ Preparation of cDNA for real-time PCR

■ Fast cDNA synthesis with integrated genomic DNA removal QuantiTect Reverse Transcription Kit 486

■ cDNA synthesis from limited amounts of RNA by whole transcriptome amplification QuantiTect Whole Transcriptome Kit 445

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

■ Genomewide, predesigned primer sets QuantiTect Primer Assays New QuantiTect Primer Assay Plates

487487

■ Ultrafast cycling on Rotor-Gene cyclers New Rotor-Gene SYBR Green Kits 489

■ Fast cycling QuantiFast SYBR Green Kits 491

■ Standard cycling with optional UNG pretreatment QuantiTect SYBR Green Kits 494

■ microRNA and mRNA detection miScript PCR System 558

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

■ Ultrafast cycling on Rotor-Gene cyclers New Rotor-Gene Probe Kits 497

■ Fast cycling QuantiFast Probe Kits 499

■ Standard cycling with optional UNG pretreatment QuantiTect Probe Kits 502

■ Viral DNA and RNA detection QuantiTect Virus Kits 505

■ SNP genotyping Type-it Fast SNP Probe PCR Kit 507

■ Methylation analysis EpiTect MethyLight PCR KitsEpiTect MethyLight Assays

163164

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

■ Ultrafast cycling on Rotor-Gene cyclers New Rotor-Gene Multiplex Kits 508

■ Fast cycling QuantiFast Multiplex PCR Kits New QuantiFast Multiplex RT-PCR Kits

510510



n Standard cycling with optional UNG pretreatment QuantiTect Multiplex Kits 513

n Viral DNA and RNA detection QuantiTect Virus Kits 505

n HRM (high-resolution melt) analysis

n Detection of gene mutations and SNPs New Type-it HRM PCR Kit 516

n Methylation analysis New EpiTect HRM PCR Kit 165

n Automated solutions

n RNA purification and RT-PCR setup in 96-well format BioRobot Universal System 62

n Reaction setup in all formats New QIAgility 67n Real-time PCR cycler

and HRM analyzer New Rotor-Gene Q 78

10.3 Cloning www.qiagen.com/PG/cloning

n Cleanup of PCR products See PCR cleanup 362

n Cloning of PCR products

n With competent cells QIAGEN PCR Cloningplus Kit 518

n Without competent cells QIAGEN PCR Cloning Kit 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.

für Seitenzahl

in DATAform




End-point PCR: applications

QIAGEN

Fast

Cycli

ng PC

R Kit

QIAGEN

Mult

iplex

PCR K

it

Type-i

t Micr

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llite P

CR K

it

Type-i

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tion D

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PCR K

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QIAGEN

Long

Rang

e PCR

Kit

HotStar

HiFi

delity

Polym

erase

Kit

QIAGEN

Taq D

NA Polym

erase

QIAGEN

Taq P

CR Co

re Kit

QIAGEN

Taq P

CR M

aster

Mix

Kit

TopT

aq D

NA Polym

erase

TopT

aq M

aster

Mix

Kit

HotStar

Taq D

NA Polym

erase

HotStar

Taq M

aster

Mix

Kit

HotStar

Taq P

lus D

NA Polym

erase

HotStar

Taq P

lus M

aster

Mix

Kit

EpiTe

ct MSP

Kit

Applications PCR

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.

PCR Assay Technologies 10.1

466 470 472 473 469 468 457 457 459 455 456 462 465 460 461 162


QIAGEN

Fast

Cyclin

g PCR

Kit

QIAGEN

Mult

iplex

PCR K

it

Type-i

t Micr

osate

llite P

CR Kit

Type-i

t Muta

tion D

etect

PCR K

it

QIAGEN

Long

Rang

e PCR

Kit

HotStar

HiFi

delity

Polym

erase

Kit

QIAGEN

Taq D

NA Polym

erase

QIAGEN

Taq P

CR Co

re Kit

QIAGEN

Taq P

CR M

aster

Mix

Kit

TopTaq

DNA Po

lymera

se

TopTaq

Mas

ter M

ix Kit

HotStar

Taq D

NA Polym

erase

HotStar

Taq M

aster

Mix

Kit

HotStar

Taq Pl

us D

NA Polym

erase

HotStar

Taq Pl

us M

aster

Mix

Kit

EpiTe

ct MSP

Kit

End-point PCR: features

Benefits Advantages PCR

Speed Ultrafast PCR n

Multiplex PCR n n n

Hot-start (5 min)

n n n n n n

PCR perform-ance

Hot-start (15 min)

n n n

Hot-start (5 min)

n n n n n n

Maximal specificity

n n n n n n n n n n

Maximal sensitivity

n n n n n n n n n n

Q-Solution n n n n n n n n n n n

Ease of use and conve-nience

Direct UA/TA cloning

n n n n n n n n n n n n n n n

PCR with gel tracking dyes

n n n n n n n n

Room-temperature setup

n n n n n n n n n n n n

Complete kit with dNTPs

n n n

Master mix with dNTPs

n n n n n n n n n

Fridge storage

n n

Page 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.

QIAGEN

Fast

Cyclin

g PCR

Kit*

QIAGEN

Mult

iplex

PCR K

it†

Type-i

t Micr

osate

llite P

CR Kit‡

Type-i

t Muta

tion D

etect

PCR K

it‡

QIAGEN

Long

Rang

e PCR

Kit§

HotStar

HiFi

delity

Polym

erase

Kit†

QIAGEN

Taq D

NA Polym

erase

†

QIAGEN

Taq P

CR Co

re Kit†

QIAGEN

Taq P

CR M

aster

Mix

Kit†

TopTaq

DNA Po

lymera

se†

TopTaq

Mas

ter M

ix Kit†

HotStar

Taq D

NA Polym

erase

†

HotStar

Taq M

aster

Mix

Kit†

HotStar

Taq Pl

us D

NA Polym

erase

†

HotStar

Taq Pl

us M

aster

Mix

Kit†

EpiTe

ct MSP

Kit‡

End-point PCR and RT-PCR 10.1

466 470 472 473 469 468 457 457 459 455 456 462 465 460 461 162



End-point RT-PCR: applications

QIAGEN

Lon

gRan

ge 2S

tep RT

-PCR K

it

QIAGEN

One

Step

RT-PC

R Kit

Omniscri

pt RT

Kit

Sens

iscrip

t RT K

it

Applications RT-PCR

Two-step RT-PCR* ■ ■ ■

One-step RT-PCR* ■

Long-range, two-step RT-PCR ■

Single-cell, one-step RT-PCR ■

Virus detection ■


* Including gene expression analysis.

QIAGEN

Lon

gRan

ge 2S

tep RT

-PCR K

it*

QIAGEN

One

Step

RT-PC

R Kit†

Omniscri

pt RT

Kit‡

Sens

iscrip

t RT K

it‡

Benefits Advantages RT-PCR

PCR and RT-PCR performance

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 ■ ■ ■


(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.

End-point RT-PCR: features


475 474 477 478

475 474 477 478



QIAGEN

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M

A

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 amplified in lanes 11–14 in Figure A were amplified under identical cycling conditions using DNA polymerases from the indicated suppliers. TopTaq DNA Polymerase (QIAGEN) showed high yields of specific PCR products regardless of fragment size.

B

QIAGEN Supplier A Supplier I Supplier T11 12 13 14 11 12 13 14 11 12 13 14 11 12 13 14

TopTaq® DNA Polymerase For highly reliable end-point PCR applications with unrivaled ease-of-use

n 4°C storage for immediate reaction setup n Convenient room-temperature handling n A single preoptimized protocol suitable for all PCR assays n 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 4°C 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.

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

Related productsAutomated PCR setup QIAgility 67

Automated DNA QIAxcel System 83fragment analysis

Product Contents Cat. no.

TopTaq DNA Polymerase (250 U)*† For 200 x 50 μl reactions‡ 200203

TopTaq DNA Polymerase (1000 U)*† For 800 x 50 μl reactions‡ 200205

TopTaq DNA Polymerase (5000 U)*† For 4000 x 50 μl reactions‡ 200207* 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 Coral-Load Concentrate, 5x Q-Solution®, and 25 mM MgCl2.




M M25°C4°C−20°C

A

10000.05000.02000.01000.0

700.0

500.0

400.0

300.0

200.0

100.0

15.0

B00

A01

A02 A03

A04

A05 A06

A07

A08

A09 A10

A11

A12

A04

B

−20°C 4°C 25°C

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 freeze–thaw cycles ■ Reliable high-yield PCR performance ■ Convenient room-temperature handling ■ Optimized protocol suitable for all PCR assays

Product description

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 4°C 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 25°C, 4°C, or –20°C.

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.

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


Automated DNA fragment analysis QIAxcel System 83


TopTaq Master Mix Kit (250 U) For 200 x 50 µl reactions* 200403* Includes 2x TopTaq Master Mix (with 250 units of TopTaq DNA Polymerase, 3 mM MgCl2, and 400 µM each dNTP),

10x CoralLoad Concentrate, and RNase-free water.

Reliable high-yield PCR independent of storage conditions. PCR was performed using TopTaq Master Mix stored at –20°C, 4°C, and 25°C for 4 months. Two human genes (a 750 bp fragment of the prp gene, and a 1200 bp fragment of the hugl gene) were amplified according to the standard optimized protocol (duplicates shown). The results show high yields of specific 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.




—15.0—194.0

—603.0

—3000.01078.0—

A07

A07

A08

A09

A10

A11

A12

A08

310.0—

M A B C M

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.

A B

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.

Taq DNA Polymerase and Taq PCR Core Kit For standard and specialized PCR applications

n QIAGEN PCR Buffer for minimal optimization n Additional ready-to-load PCR buffer for faster handling n Q-Solution for amplification of GC-rich templates n 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:

n General PCR n RT-PCR n Differential display n PCR-based DNA fingerprinting (VNTR, STR, and RAPD)



Molecular weight markers GelPilot Molecular Weight Markers 582




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


Taq DNA Polymerase (250 U)* For 200 x 50 μl reactions† 201203



Taq DNA Polymerase (25,000 U)* For 20,000 x 50 μl reactions† 201209

Taq PCR Core Kit (250 U) For 200 x 50 μl reactions†‡ 201223

Taq PCR Core Kit (1000 U) For 800 x 50 μl reactions†‡ 201225

* For dNTPs, see page 467. † 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).



M 1C 0 20 50 copies

– 188 bp

Reproducible PCR. A fragment of the hepatitis B surface antigen gene (gene S) was amplified from 10, 20, and 50 copies of target template, using the Taq PCR Master Mix Kit. Five parallel amplifications 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.

Taq PCR Master Mix Kit Premixed solution for convenient PCR setup

n Easy reaction setup n Fewer pipetting steps n 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:

n General PCR n RT-PCR n Differential display n PCR-based DNA fingerprinting (VNTR, STR, and RAPD)

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





Taq PCR Master Mix Kit (250 U) For 200 x 50 μl reactions* 201443

Taq PCR Master Mix Kit (1000 U) For 800 x 50 μl reactions* 201445* Includes Taq PCR Master Mix (with 1.5 mM MgCl2 and 200 μM each dNTP), containing 250 or 1000 units of Taq DNA Polymerase,

and RNase-free water.




M MHotS

tarTa

q Plus

HotStar

Taq

Supp

lier I

Supp

lier A

ll

Taq DNA

Polym

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Supp

lier R

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 amplified in 35 PCR cycles. M: markers.

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 (seepage 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).

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





HotStarTaq Plus DNA Polymerase (250 U)*† For 200 x 50 μl reactions‡ 203603

HotStarTaq Plus DNA Polymerase (1000 U)* For 800 x 50 μl reactions‡ 203605

HotStarTaq Plus DNA Polymerase (5000 U)*† For 4000 x 50 μl reactions‡ 203607

HotStarTaq Plus DNA Polymerase (25,000 U)* For 20,000 x 50 μl reactions‡ 203609

* For dNTPs, see page 467. † Polymerase supplied in single tube. ‡ Includes 250, 1000, 5000, or 25,000 units of HotStarTaq PlusDNA Polymerase, 10x PCR Buffer (contains 15 mM MgCl2), 10x CoralLoad PCR Buffer (contains 15 mM MgCl2), 5x Q-Solution, and 25 mM MgCl2.



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.

HotStarTaq Plus Master Mix Kit (2500). Bulk-size kit containing single tube of 2.5 ml master mix.

HotStarTaq Plus Master Mix Kit For fast and highly specific amplification in all applications

n Fast 5-minute enzyme activation time n Fewer pipetting steps reduce the risk of contamination n Higher PCR specificity and reduced nonspecific amplification n 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).




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

A B


HotStarTaq Plus Master Mix Kit (250)* For 250 x 20 μl reactions† 203643

HotStarTaq Plus Master Mix Kit (1000) For 1000 x 20 μl reactions† 203645

HotStarTaq Plus Master Mix Kit (2500)* For 2500 x 20 μl reactions† 203646* Master mix supplied in single tube.† Includes HotStarTaq Plus Master Mix (with 1.5 mM MgCl2 and 200 μM each dNTP), containing 250, 1000, or 2500 units

HotStarTaq Plus DNA Polymerase, 10x CoralLoad Concentrate, and RNase-free water.




HotStarTaq DNA Polymerase For highly specific amplification in any PCR application

Highly specific PCR ■

Reduced nonspecific amplification ■

Easy handling and room-temperature setup ■

Add primersand template

Distribute

Amplification

Reaction setupat room temperature

C04

C05

C06

C07

C08

C09

C04

C05

C06

C07

C08

C09

3000.01200.0

500.0

300.0

Manual QIAgility

p53

p53

HUGLHUGL

NTCNTCNTCNTC

100.0

5000.02000.0

600.0400.0

200.0

15.0

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 specific amplification of both p53 (400 bp) and HUGL (1200 bp). NTC: no template control.

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, primer–dimers, 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 primer–dimers at low temperatures. HotStarTaq DNA Polymerase is activated by a 15-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs.



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 primer–template systems and maintains high primer annealing specificity in each PCR cycle. This novel buffer is provided with all QIAGEN PCR kits (pages 455–475) 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).


Automated DNA fragment analysis QIAxcel System 83

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


B H

H

Pñ

K+ NH3 + H+

DestabilizationNH4

+Stabilization Stabilization

Pñ Pñ

Pñ

B B

B

K+

K+

K+

H Strong hydrogen band(e.g., between A and T nucleotides)

H Weak hydrogen band(e.g., between C 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 specific to nonspecific primer–template binding over a wide temperature range.




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

Special feature Description Recommended kit Page

Parallel amplification of multiple products

Multiplex PCR ■ Type-it Mutation Detect PCR Kit

■ Type-it Microsatellite PCR Kit

■ QIAGEN Multiplex PCR Kit

473

472

470

Amplification of difficult genomic loci

Highly specific hot-start PCR and HRM analysis

■ Type-it HRM PCR Kit 516

Amplification of difficult SNP loci Probe-based real-time PCR ■ Type-it Fast SNP Probe PCR Kit 507High-throughput/fast PCR Fast-cycling PCR ■ QIAGEN Fast Cycling PCR Kit 466Increased sensitivity for single-cell PCR

Highly specific hot-start PCR ■ HotStarTaq Master Mix Kit

■ HotStarTaq Plus DNA Polymerase

■ HotStarTaq Plus Master Mix Kit

465

460

461

Sequence accuracy High-fidelity PCR ■ HotStar HiFidelity Polymerase Kit 468Amplification of long PCR products

Long-range PCR ■ QIAGEN LongRange PCR Kit 469

Detection of methylated DNA Methylation-specific PCR

Methylation analysis using HRM analysis■ EpiTect MSP Kit

■ EpiTect HRM PCR Kit

162

165

Increased sensitivity One-step RT-PCR

Long-range two-step RT-PCR■ QIAGEN OneStep RT-PCR Kit

■ QIAGEN LongRange 2Step RT-PCR Kit

474

475

Standard PCR End-point PCR ■ TopTaq DNA Polymerase

■ TopTaq Master Mix Kit

■ Taq DNA Polymerase

■ Taq PCR Core Kit

■ Taq PCR Master Mix Kit

455

456

457

457

459

Additional kits for dedicated applications


HotStarTaq DNA Polymerase (250 U)* For 200 x 50 μl reactions† 203203

HotStarTaq DNA Polymerase (1000 U) For 800 x 50 μl reactions† 203205

HotStarTaq DNA Polymerase (5000 U)* For 4000 x 50 μl reactions† 203207

HotStarTaq DNA Polymerase (25,000 U) For 20,000 x 50 μl reactions† 203209

* Polymerase supplied in single tube.† 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.



HotStarTaq Master Mix Kit.

HotStarTaq Master Mix Kit Premixed solution for high PCR specificity

n Easy reaction setup at room temperature n Ready to use with fewer pipetting steps n 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).

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





HotStarTaq Master Mix Kit (250 U)* For 100 x 50 μl reactions† 203443

HotStarTaq Master Mix Kit (1000 U) For 400 x 50 μl reactions† 203445

HotStarTaq Master Mix Kit (2500 U)* For 1000 x 50 μl reactions† 203446* Master mix supplied in single tube.† Includes HotStarTaq Master Mix (with 1.5 mM MgCl2 and 200 μM each dNTP), containing 250, 1000, or 2500 units of HotStarTaq

DNA Polymerase, and RNase-free water.




Fast CyclingProcedure

Standard CyclingProcedure

Enzymeactivation

35 cycles

Final extension

35 cycles

Final extension

Total timearound 20 minutes

Total time>1 hour

Enzymeactivation

Significant Time Savings QIAGEN Fast Cycling PCR Kit For ultrafast and specific PCR on any thermal cycler

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 PolymeraseNo need to redesign primers for fast PCR ■

Optional ready-to-load PCR dye for easier handling ■

Product description

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

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.

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

Related products

Automated PCR setup QIAgility 67



QIAGEN Fast Cycling PCR Kit (200)* For 200 x 20 μl reactions† 203743

QIAGEN Fast Cycling PCR Kit (1000) For 1000 x 20 μl reactions† 203745

* Master mix supplied in single tube. † Includes 2x QIAGEN Fast Cycling PCR Master Mix (with optimized MgCl2 concentration and 200 μM each dNTP), containing HotStarTaq Plus DNA Polymerase, 10x CoralLoad Dye, 5x Q-Solution, and RNase-free water.



Sample type Application

DNA Standard PCRHigh-fidelity PCRUltrafast PCRLong-range PCRMultiplex PCRGenotypingSingle-cell PCRMethylation-specific PCR (MSP)Nested PCR

RNA/cDNA

Two-step RT-PCRMultiplex, two-step PT-PCRSingle-cell, two-step PT-PCR

Ideal for all PCR and RT-PCR applications

dNTP Set, PCR Grade.

dNTP Mix, PCR Grade can be used in a variety of sensitive PCR techniques.

dNTP Set, PCR Grade and dNTP Mix, PCR Grade For sensitive and reproducible PCR and RT-PCR

n Individual dNTPs or premixed ready-to-use solution n Highly pure n 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.

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


dNTP Mix, PCR Grade (200 μl) For 200 x 50 μl reactions* 201900

dNTP Mix, PCR Grade (800 μl) For 800 x 50 μl reactions* 201901

dNTP Set, PCR Grade, 4 x 100 μl For 1000 x 50 μl reactions† 201912

dNTP Set, PCR Grade, 4 x 250 μl For 2500 x 50 μl reactions† 201913* Mix containing 10 mM each of dATP, dCTP, dGTP, and dTTP. † Separate aliquots of 100 mM each dATP, dCTP, dGTP, and dTTP.




QIAGEN

Supp

lier S

Supp

lier I

*

Supp

lier I

*

Supp

lier R

M 10 1 10 1 10 1 10 1 10 1 ng

Highly sensitive and reliable PCR. PCR was carried out using HotStar HiFidelity DNA Polymerase (QIAGEN) and 4 high-fidelity PCR enzymes from the indicated suppliers. A 2.3 kb fragment of the human IL9R gene was amplified from genomic DNA in 40 PCR cycles. M: markers. * Two different high-fidelity enzymes from Supplier I.

100

80

60

40

20

0Clon

ing

effic

ienc

y (%

)

TA cloning UA cloning

Taq DNAPolymeraseHotStarHiFidelity

Efficient TA/UA cloning. A 955 bp PCR fragment was amplified from 100 ng human genomic DNA using the HotStar HiFidelity Polymerase Kit and standard Taq DNA Polymerase. 4 μl of each PCR product was cloned using either a commercially available TA or UA cloning kit. Blue/whitescreening was used to determine the cloning efficiency.

HotStar HiFidelity Polymerase Kit For highly sensitive and reliable high-fidelity hot-start PCR

High sensitivity and specificity ■

Unique UA/TA cloning feature ■

10-fold higher fidelity than ■ Taq DNA PolymeraseRoom-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.

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





HotStar HiFidelity Polymerase Kit (100 U) For 40 x 50 μl reactions* 202602

HotStar HiFidelity Polymerase Kit (1000 U) For 400 x 50 μl reactions* 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.



M QIAGEN

Supp

lier R

Supp

lier T

M QIAGEN

Supp

lier R

Supp

lier T

– 27 kb– 24 kb

High yields of very long PCR products. 27 kb and 24 kb PCR products were amplified from complex DNA using the QIAGEN LongRange PCR Kit and 2 long-range PCR kits from the indicated suppliers according to the manufacturer’s instructions. The QIAGEN LongRange PCR Kit provided much higher yields of specific product than the other kits tested.

QIAGEN LongRange PCR Kit For sensitive and accurate long-range PCR

n Amplification of extremely long PCR products (up to 40 kb) n Low error rates, ensured by high-fidelity enzyme n Minimal PCR optimization due to unique buffer system n 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.

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




QIAGEN LongRange PCR Kit (20) For 20 x 50 μl reactions* 206401

QIAGEN LongRange PCR Kit (100) For 100 x 50 μl reactions* 206402

QIAGEN LongRange PCR Kit (250) For 250 x 50 μl reactions* 206403* Includes LongRange PCR Enzyme Mix (40, 200, or 500 units), LongRange PCR Buffer, 5x Q-Solution, RNase-free water, and 10 mM

dNTPs.




— 955 bp

— 99 bp

Supplier I QIAGEN

M 1.5 2.5 3.5 Std mM Mg2+

Successful 19-plex PCR without optimization. Multiplex PCR of 19 targets (99–955 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 amplification of all 19 fragments in parallel without the need for optimization. M: markers.

QIAGEN Multiplex PCR Kit For highly specific and sensitive multiplex PCR without optimization

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

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.

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





QIAGEN Multiplex PCR Kit (100) For 100 x 50 μl multiplex PCRs* 206143

QIAGEN Multiplex PCR Kit (1000)† For 1000 x 50 μl multiplex PCRs* 206145

* Includes QIAGEN Multiplex PCR Master Mix (containing HotStarTaq DNA Polymerase, Multiplex PCR Buffer, MgCl2 [final concentration 3 mM], and dNTPs), 5x Q-Solution, and RNase-free water.

† Master mix supplied in single tube.



New PyroMark® PCR Kit For PCR amplification of template DNA optimized for Pyrosequencing® analysis

n Specific amplification of bisulfite-converted DNA n Specific amplification of genomic DNA from various sources n Optimized for successful Pyrosequencing analysis n Consistently high yields of PCR product n 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.


PyroMark PCR Kit (200) For 200 reactions* 978703

PyroMark PCR Kit (800) For 800 reactions* 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

Related productsBisulfite conversion EpiTect Bisulfite Kits 159of DNA

Pyrosequencing PyroMarkanalysis Systems 72

GGGGE

B8: TACTCRTAAATACRACRCTTATATTCTTAAAAAAAATTTTAC

S CCCCCC AAAAAAAAA TTTTTT252015105

T

130120110100

908070605040302010

0

54% 57% 48%

125100

755025

0

GGGGE S CCCCCC AAAAAAAAA TTTTTT

252015105

T

B7: TACTCRTAAATACRACRCTTATATTCTTAAAAAAAATTTTAC50% 58% 45%

PyroMark PCR Kit

Standard PCR

PCR amplification optimized for Pyrosequencing analysis. The PyroMark PCR Kit ensures greater amplification efficiency with high specificity. This leads to higher peaks and more reliable Pyrosequencing results compared to standard PCR.




80 130 160 200 240 280 320

5000

4000

3000

2000

1000

0

FAM Channel

80 130 160 200 240 280 320

5000

4000

3000

2000

1000

0

NED Channel

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.

Type-it® Microsatellite PCR Kit For fast and reliable multiplex PCR analysis of microsatellite loci

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.

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.


Type-it Microsatellite PCR Kit (70) For 70 x 25 μl reactions* 206241

Type-it Microsatellite PCR Kit (200) For 200 x 25 μl reactions* 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

Related products




B

Type-it MM

MM 0 ng 25

ng0.2

5 ng

25 pg M 0

ng 25 ng

0.25

ng

25 pg

Supplier I

357bp

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.

Type-it Mutation Detect PCR Kit For accurate and reliable multiplex PCR analysis of mutations

n Successful and reproducible analysis of multiple mutations n Multiplex PCR assay development without optimization n Specific and sensitive coamplification of all fragments n 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).

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

Related productsAutomated DNA QIAxcel System 83fragment analysis


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)


Type-it Mutation Detect PCR Kit (70) For 70 x 25 μl reactions* 206341

Type-it Mutation Detect PCR Kit (200) For 200 x 25 μl reactions* 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.




M 10010 1 0 10010 1 0 10010 1 0 10010 1 0 pg

QIAGEN Supplier R Supplier I Supplier BIV

Efficient, sensitive RT-PCR. One-step RT-PCR was carried out using the indicated amounts of total RNA from HeLa cells and primers specific for β-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 specific results even with low amounts of template. M: 100 bp ladder.

M 22,0

00

2200

220

22 2 copies

Effective detection of viral RNA. A 336 bp fragment of F-gene mRNA was reverse-transcribed and amplified 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.)

QIAGEN OneStep RT-PCR Kit For highly sensitive and successful one-step RT-PCR

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.

Related productsAutomated DNA QIAxcel System 83fragment analysis

RNA purification RNeasy Kits 409



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


QIAGEN OneStep RT-PCR Kit (25) For 25 x 50 μl reactions* 210210

QIAGEN OneStep RT-PCR Kit (100) For 100 x 50 μl reactions* 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.



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 HActivity

RNase H Activity Quencher

RNA

Polymerase ActivityPolymerase Activity

X

X

X

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 hybrid–binding site of the reverse transcriptase, preventing degradation of the RNA during synthesis. This results in reduced RNase H activity and the amplification of full-length cDNA up to 12.5 kb.

QIAGEN LongRange 2Step RT-PCR Kit For sensitive and accurate long-range two-step RT-PCR

n Dedicated solution for amplification of long transcripts n High-yield amplification of up to 12.5 kb cDNA n Low error rates and extended read length n Minimal PCR optimization due to unique buffer system n 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.

M Tubuli

n

TNFR

1

MhTSF

Dynein

12.3 kb –5.3 kb –

13 kb –

500 bp –

Related productsAutomated DNA fragment QIAxcel System 83analysis







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


QIAGEN LongRange For 10 x 20 μl RT reactions and 2059202Step RT-PCR Kit (20) 20 x 50 μl PCR reactions*

QIAGEN LongRange For 50 x 20 μl RT reactions and 2059222Step RT-PCR Kit (100) 100 x 50 μl PCR reactions*

* Includes reagents for reverse transcription and PCR. For RT step: LongRange Reverse Transcription Enzyme, buffer, dNTPs, oligo-dT, RNase inhibitor, RNase-free water. For PCR step: QIAGEN LongRange PCR Kit (see page 469).



AMV(Supplier P)

AMV RNase H–(Supplier I)

MMLV(Supplier I)

MMLV RNase H–(Supplier I)

1000 500 250 125 62 31 ng RNA

Omniscript(QIAGEN)

Superior sensitivity and dynamic range of Omniscript RT. Reverse transcription was carried out with different reverse transcriptases according to suppliers’ specifications, using the indicated amounts of total RNA from HeLa cells. 1/20 of the reverse-transcription reaction was used in a 25-cycle PCR amplification with QIAGEN Taq DNA Polymerase. A 1.7 kb b-actin fragment was amplified.

Omniscript RT Kit For reverse transcription of 50 ng to 2 µg RNA per reaction

n High cDNA yields due to high-affinity enzyme n Sensitive detection of as few as 10 copies of template n Fast and easy procedure with no tedious pipetting steps n 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 μg 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: n Standard RT and RT-PCR n Quantitative, real-time RT-PCR n Primer extension and RACE analysis n Synthesis of double-stranded cDNA for cloning

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

Related productsSample disruption TissueLyser LT 41 TissueLyser II 42


End-point PCR HotStarTaq DNA Polymerase 462


Omniscript RT Kit (10) For 10 x 20 μl reactions* 205110

Omniscript RT Kit (50) For 50 x 20 μl reactions* 205111

Omniscript RT Kit (200) For 200 x 20 μl reactions* 205113* Includes Omniscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix (5 nM of each dNTP), and RNase-free water.




-Actin

NF- B

Inositolreceptor

Dystrophin

– 210 bp

– 460 bp

– 240 bp

– 360 bp

500 50 10 1 0 cells

RT-PCR with RNA corresponding to 1 cell. Total RNA was purified 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 specific for genes encoding β-actin, NF-κB, inositol-1,4, 5-triphosphate receptor, or dystrophin. Sizes of the amplicons are as indicated.

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

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 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 ■

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

Related productsSample disruption TissueLyser LT 41 TissueLyser II 42


End-point PCR HotStarTaq DNA Polymerase 462


Sensiscript RT Kit (50) For 50 x 20 μl reactions* 205211

Sensiscript RT Kit (200) For 200 x 20 μl reactions* 205213

* Includes Sensiscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix (5 nM of each dNTP), and RNase-free water.


Real-time PCR and RT-PCR 10.2

Rotor

-Gen

e SYB

R Gree

n PCR

Kit

Rotor

-Gen

e SYB

R Gree

n RT-P

CR Kit

Rotor

-Gen

e Prob

e PCR

Kit

Rotor

-Gen

e Prob

e RT-P

CR Kit

Rotor

-Gen

e Mult

iplex

PCR K

it

Rotor

-Gen

e Mult

iplex

RT-PC

R Kit

Detection Template Procedure Real-time PCR/RT-PCR

SYBR Green Genomic DNA PCR* n

cDNA Two-step RT-PCR* n †‡

RNA One-step RT-PCR* n †

Cells Two-step RT-PCR* n †§

Probe Genomic DNA PCR* n

cDNA Two-step RT-PCR* n ‡

RNA One-step RT-PCR* n

Cells Two-step RT-PCR* n §

Multiplex Genomic DNA PCR* n

cDNA Two-step RT-PCR* n ‡

RNA One-step RT-PCR* n

Cells Two-step RT-PCR* n §

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).† 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.

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

489 489 497 497 508 508




Real-time PCR and RT-PCR: fast cycling

Quanti

Fast

SYBR

Gree

n PCR

Kit

Quanti

Fast

SYBR

Gree

n RT-P

CR K

it

Quanti

Fast

Probe

PCR K

it

Quanti

Fast

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PCR +

ROX V

ial K

it

Type-i

t Fas

t SNP P

robe

PCR K

it

Quanti

Fast

Probe

RT-PC

R Kit

Quanti

Fast

Probe

RT-PC

R +RO

X Vial

Kit

Quanti

Fast

Multipl

ex PC

R Kit

Quanti

Fast

Multipl

ex PC

R +R K

it

Quanti

Fast

Multipl

ex RT

-PCR K

it

Quanti

Fast

Multipl

ex RT

-PCR +

R Kit

Type-i

t HRM

PCR K

it

EpiTe

ct HRM

PCR K

it

Detection Template Procedure ROX dye Real-time PCR/RT-PCR

SYBR Green

Genomic DNA

PCR* In master mix■

cDNA Two-step RT-PCR*

In master mix■ †‡

RNA One-step RT-PCR*

In master mix■ †

Probe Genomic DNA

PCR* In master mix ■ ■ §

In separate tube ■


In master mix ■ ‡

In separate tube ■ ‡


In master mix ■


Multiplex Genomic DNA

PCR* In master mix ■



In master mix ■ ‡

In separate tube ■ ‡


In master mix ■


HRM Genomic DNA

PCR* Not included■

Bisulfite-converted DNA

PCR* Not included■


* 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.

491 491 499 499 507 499 499 510 510 510 510 516 165



Quanti

Fast

SYBR

Gree

n PCR

Kit

Quanti

Fast

SYBR

Gree

n RT-P

CR Kit

Quanti

Fast

Probe

PCR K

it

Quanti

Fast

Probe

PCR +

ROX V

ial Kit

Type-i

t Fas

t SNP P

robe P

CR Kit

Quanti

Fast

Probe

RT-PC

R Kit

Quanti

Fast

Probe

RT-PC

R +RO

X Vial

Kit

Quanti

Fast

Multipl

ex PC

R Kit

Quanti

Fast

Multipl

ex PC

R +R K

it

Quanti

Fast

Multipl

ex RT

-PCR K

it

Quanti

Fast

Multipl

ex RT

-PCR +

R Kit

Type-i

t HRM

PCR K

it

EpiTe

ct HRM

PCR K

it

Quanti

Tect S

YBR G

reen P

CR Kit

Quanti

Tect S

YBR G

reen R

T-PCR

Kit

Quanti

Tect P

robe P

CR Kit

Quanti

Tect P

robe R

T-PCR

Kit

Quanti

Tect M

ultipl

ex PC

R Kit

Quanti

Tect M

ultipl

ex PC

R NoR

OX Kit

Quanti

Tect M

ultipl

ex RT

-PCR K

it

Quanti

Tect M

ultipl

ex RT

-PCR N

R Kit

Quanti

Tect V

irus K

it

Quanti

Tect V

irus +

ROX V

ial Kit

EpiTe

ct Meth

yLigh

t PCR

Kit

EpiTe

ct Meth

yLigh

t PCR

+ROX V

ial Kit

Detection Template Procedure ROX dye Real-time PCR/RT-PCR

SYBR Green

Genomic DNA

PCR* In master mixn ‡


In master mixn ‡§¶


In master mixn §

Probe Genomic DNA

PCR* In master mixn ‡


In master mixn ‡ ¶


In master mixn

Viral DNA and RNA

PCR and one-step RT-PCR†

In master mix n

In separate tube n

Bisulfite-converted DNA

PCR† In master mix n

In separate tube n

Multiplex Genomic DNA

PCR* In master mix n

Not included n


In master mix n ¶

Not included n ¶


In master mix n

Not included n

Viral DNA and RNA

PCR and one-step RT-PCR†

In master mix n

In separate tube n

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 amaster 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).

Real-time PCR and RT-PCR: standard cycling

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




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

Quanti

Tect S

YBR G

reen P

CR K

it

Quanti

Tect P

robe

PCR K

it

Quanti

Tect M

ultipl

ex PC

R Kit

Quanti

Tect M

ultipl

ex PC

R NoR

OX Kit

Quanti

Fast

SYBR

Gree

n PCR

Kit

Quanti

Fast

Probe

PCR K

it

Quanti

Fast

Probe

PCR +

ROX V

ial K

it

Quanti

Fast

Multipl

ex PC

R Kit

Quanti

Fast

Multipl

ex PC

R +R K

it

FastL

ane C

ell SY

BR G

reen K

it

FastL

ane C

ell Pr

obe K

it

FastL

ane C

ell M

ultipl

ex K

it

FastL

ane C

ell M

ultipl

ex N

R Kit

Detection Procedure Cycling ROX dye With FastLane Cell cDNA Kit (page 483) Complete kits

SYBR Green

Two-step RT-PCR*

Standard‡ In master mix ■ ¶

Fast§ In master mix ■ ¶

One-step RT-PCR†

Standard‡ In master mix ■ ¶

Probe Two-step RT-PCR*

Standard‡ In master mix

■

Fast§ In master mix ■

In separate tube

■

One-step RT-PCR†

Standard‡ In master mix

■

Multiplex Two-step RT-PCR*

Standard‡ In master mix ■

Not included

■

Fast§ In master mix ■

In separate tube

■

One-step RT-PCR†

Standard‡ In master mix ■

Not included

■


* 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).

494 502 513 491 499 499 510 510 484 484 484 484513


Quanti

Tect S

YBR G

reen P

CR Kit

Quanti

Tect P

robe P

CR Kit

Quanti

Tect M

ultipl

ex PC

R Kit

Quanti

Tect M

ultipl

ex PC

R NoR

OX Kit

Quanti

Fast

SYBR

Gree

n PCR

Kit

Quanti

Fast

Probe

PCR K

it

Quanti

Fast

Probe

PCR +

ROX V

ial Kit

Quanti

Fast

Multipl

ex PC

R Kit

Quanti

Fast

Multipl

ex PC

R +R K

it

FastL

ane C

ell SY

BR G

reen K

it

FastL

ane C

ell Pr

obe K

it

FastL

ane C

ell M

ultipl

ex Kit

FastL

ane C

ell M

ultipl

ex N

R Kit


A

B

160

120

80

40

0Rela

tive

expr

essi

on o

f CDC

2 (%

)

S1 S2 S3 NS UT

160

120

80

40

0Rela

tive

expr

essi

on o

f CDC

2 (%

)

S1 S2 S3 NS UT

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.

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

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

Product description

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:

n Validation of siRNA-mediated gene knockdown n Evaluation of drug effects n Detection of gene regulation

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

Related products

Gene silencing FlexiTube siRNA 543

Ultrafast real-time PCR Rotor-Gene Kits 479

Fast real-time PCR QuantiFast Kits 480

Real-time PCR with QuantiTect Kits 481UNG pretreatment


FastLane Cell cDNA Kit (50) For 50 x 20 μl reactions* 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 sufficient 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).




n = 384Mean C = 25.24CV = 1.97%

T

Highly reproducible analysis of a 384-well plate. Real-time one-step RT-PCR analysis of the expression of NFκB1 (nuclear transcription factor) using the FastLane Cell Probe Kit gave reproducible CT values from a 384-well plate of HeLa cells.

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.



HeLa HepG2 Cos

Sensitive detection without RNA purification. Two low-abundance transcripts, INPP5D (a phosphatase) and SRF (serum response factor), were quantified 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.)

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


FastLane Cell SYBR Green Kit (200)* For 200 x 50 μl reactions† 216213

FastLane Cell Probe Kit (200)‡ For 200 x 50 μl reactions† 216413

FastLane Cell Multiplex Kit (200)§ For 200 x 50 μl reactions† 216513

FastLane Cell Multiplex NR Kit (200)¶ For 200 x 50 μl reactions† 216713* 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 sufficient reagent for

real-time one-step RT-PCR from 2 x 96-well cell culture plates (50 μl PCR volume) or 1 x 384-well cell culture plate (25 μl PCR volume).‡ For real-time one-step RT-PCR using sequence-specific probes.§ For multiplex, real-time one-step RT-PCR. Recommended for cyclers that require ROX dye for fluorescence 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 fluorescence normalization

(e.g., Rotor-Gene cyclers and instruments from Bio-Rad, Cepheid, Eppendorf, Roche, and Stratagene).

Related productsGene silencing FlexiTube siRNA 543 FlexiPlate siRNA 547

Transfection HiPerFect Transfection Reagent 569

Gene expression assays QuantiTect Primer Assays 487




Cycle10

Rn 140

290

240

190

90

40

–1020 30 40 50

QIAGENSupplier ISupplier AII

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 μg 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 efficiently reverse transcribed and sensitively detected in real-time PCR.

QuantiTect Reverse Transcription Kit For fast cDNA synthesis for sensitive real-time two-step RT-PCR

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 ■

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.

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

Related productsRNA purification RNeasy Kits 409

Ultrafast real-time PCR Rotor-Gene Kits 479

Fast real-time PCR QuantiFast Kits 480

Real-time PCR with QuantiTect Kits 481UNG pretreatment


QuantiTect Reverse Transcription Kit (10) For 10 x 20 μl reactions* 205310



* Includes gDNA Wipeout Buffer, Quantiscript Reverse Transcriptase, 5x Quantiscript RT Buffer (with dNTPs), RT Primer Mix, and RNase-free water.



800700600500400300200100

0-100

PCR

base

line

sub

tract

edCF

RFU

Cycle10 15 20 25 30 35 40 450

100 ng10 ng1 ng0.1 ng

5

A

100

80

60

40

20

0

-20–

d(RF

U) /

dT

Temperature (°C) 81 84 87 9069 787566 72

B

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 flat plots). B Melting curve analysis, demonstrating high PCR specificity.

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

n Guaranteed results with genomewide, predesigned primers n ~100% PCR efficiencies for reliable relative quantification n High sensitivity and specificity n Accurate quantification over a wide linear range n 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.




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 quantified 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 specificity, as indicated by the single peaks in melting curve analysis.

Related products

Reaction setup QIAgility 67

Real-time cycler Rotor-Gene Q 78

cDNA synthesis QuantiTect Reverse Transcription Kit 486

QuantiTect Whole Transcriptome Kit 445

FastLane Cell cDNA Kit 483

Real-time RT-PCR Rotor-Gene SYBR Green Kits 489

QuantiFast SYBR Green Kits 491

QuantiTect SYBR Green Kits 494

FastLane Cell SYBR Green Kit 484

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


QuantiTect Primer Assay (200) For 200 x 50 μl reactions; Varies primer set in single tube* New QuantiTect Primer Assay 96 Plate (20) For 20 x 50 μl reactions; Varies primer sets in 96-well plate*†

New QuantiTect Primer Assay 96 Plate (100) For 100 x 50 μl reactions; Varies primer sets in 96-well plate*†

New QuantiTect Primer Assay 384 Plate (20) For 20 x 50 μl reactions; Varies primer sets in 384-well plate*‡

* 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.

Rn

7.06.05.04.03.02.01.0

0

MAPK1, CT = 25.1 IL6, CT = 30.7

Cycle10 15 20 25 30 35 405

SYBR Green-based assay (QIAGEN)A

MAPK1IL6

4.0

3.0

2.0

1.0

0Der

ivat

ive

repo

rter

(-Rn)

Temperature (°C)65 75 85 9590807060

Rn

2.5

2.0

1.5

1.0

0.5

0

MAPK1, CT = 29.5 IL6, CT = 33.7

Cycle10 15 20 25 30 35 405

B Probe-based assay (Supplier AII)


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 amplification of specific 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 coamplification of nonspecific products was observed (melting curve shown in inset).

New Rotor-Gene SYBR Green Kits For ultrafast, precise real-time PCR and RT-PCR using SYBR Green I

n Ultrafast, reliable results on Rotor-Gene cyclers n Specific detection of even low copy numbers n Accurate detection of a wide range of template amounts n Specially formulated master mix for fast cycling n 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 2–8°C. 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.


Amplification plot (QIAGEN)

Nor

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ence

0

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Amplification plot (Supplier R)B

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Temperature (°C)7060 80 90 100




QIAGEN Supplier AII CT Mean deviation CT Mean deviation

BAX (BCL2-associated X protein) 24.84 0.05 29.57 0.46

BCL2 (apoptosis gene) 26.96 0.05 32.83 0.29

MYC (proto-oncogene) 28.42 0.21 35.26 0.72

β-Actin (housekeeping gene) 20.24 0.03 24.39 0.12

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 β-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.


Rotor-Gene SYBR Green PCR Kit (80) For 80 x 25 μl reactions* 204072

Rotor-Gene SYBR Green PCR Kit (400) For 400 x 25 μl reactions* 204074

Rotor-Gene SYBR Green RT-PCR Kit (400) For 400 x 25 μl reactions† 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.

Related products



DNA purification QIAamp DNA Blood Kits 267


cDNA synthesis QuantiTect Reverse Transcription Kit 486 QuantiTect Whole Transcriptome Kit 445 FastLane Cell cDNA Kit 483

Predesigned primer sets QuantiTect Primer Assays 487

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

Superior performance in RT-PCR with SYBR Green



Copies

— 1000 — 100 — 50 — 25 — 12 — 6 — 3 — 1

13

6

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-specific 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.

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 quantification 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).

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

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

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 2–8°C. For real-time one-step RT-PCR applications, an optimized mix of reverse transcriptases provides fast and efficient cDNA synthesis.




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 specific to nonspecific primer–template binding over a wide temperature range.

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.

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 first 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 first denatured and the tertiary complex is then formed by the simultaneous binding of primer and polymerase, allowing extension to start within seconds.

3'

5'

5'

3'

5'

3'

5' 3' Highly specific annealing

Fast cycling

Standard cycling A

B

Q-Bond molecule PrimerTemplate DNATaq DNA polymerase

H

NH3 + H+K+

NH4+Stabilization Destabilization

K+

P_

Template Primer

P_

BB



Special feature Description Recommended kit Page

Fast real-time PCR using probes Master mix containing Q-Bond ■ QuantiFast Probe Kits 499

Ultrafast real-time PCR using probes

Master mix containing Q-Bond, for Rotor-Gene cyclers

■ Rotor-Gene Probe Kits 497

Fast multiplex real-time PCR using probes

Master mix containing Q-Bond and Factor MP

■ QuantiFast Multiplex Kits 510

Ultrafast multiplex real-time PCR using probes

Master mix containing Q-Bond and Factor MP, for Rotor-Gene cyclers

■ Rotor-Gene Multiplex Kits 508

Detection of microRNAs Complete system for miRNA/mRNA detection

■ miScript PCR System 558

Sensitive detection of viruses Highly concentrated 5x master mix ■ QuantiTect Virus Kits 505

Detection of SNPs and mutations

Master mix developed for reliable and clear allelic discrimination

■ Type-it Fast SNP Probe PCR Kit 507

Quantification of methylation status

Master mix for reliable quantification of methylation status

■ EpiTect MethyLight PCR Kits 163

Alternative real-time PCR kits

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.

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


QuantiFast SYBR Green PCR Kit (80) For 80 x 25 μl reactions* 204052

QuantiFast SYBR Green PCR Kit (400) For 400 x 25 μl reactions* 204054

QuantiFast SYBR Green PCR Kit (2000) For 2000 x 25 μl reactions*† 204056

QuantiFast SYBR Green RT-PCR Kit (400) For 400 x 25 μl reactions‡ 204154

QuantiFast SYBR Green RT-PCR Kit (2000) For 2000 x 25 μl reactions†‡ 204156

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

Related products








QuantiTect SYBR Green PCR Kit

Supplier AII

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.

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

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 2–8°C. 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).



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)

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 on the LightCycler 2.0 using the QuantiTect Primer Assay for human MAPK14 and the indicated kits.

Nonspecificproducts

QIAGENA Supplier RB

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 specific for Myc (a protooncogene). Melting curve analysis (see insets) revealed higher PCR specificity with the QuantiTect Kit than with the kit from Supplier R.

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.




Related products








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


QuantiTect SYBR Green PCR Kit (40) For 40 x 50 μl reactions* 204141

QuantiTect SYBR Green PCR Kit (200) For 200 x 50 μl reactions* 204143

QuantiTect SYBR Green PCR Kit (1000) For 1000 x 50 μl reactions*† 204145

QuantiTect SYBR Green PCR +UNG Kit (200) For 200 x 50 μl reactions‡ 204163

QuantiTect SYBR Green RT-PCR Kit (200) For 200 x 50 μl reactions§ 204243

QuantiTect SYBR Green RT-PCR Kit (1000) For 1000 x 50 μl reactions†§ 204245

* 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.



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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%.

New Rotor-Gene Probe Kits For ultrafast, precise real-time PCR and RT-PCR using sequence-specific probes

n Ultrafast, reliable results on Rotor-Gene cyclers n Sensitive detection of even low copy numbers n Accurate detection of a wide range of template amounts n Specially formulated master mix for fast cycling

Product description

Rotor-Gene Probe Kits are dedicated for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly sensitive quantification in 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.

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 2–8°C. 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.




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.

Related productsReaction setup QIAgility 67





Virus detection QuantiTect Virus Kits 505


Rotor-Gene Probe PCR Kit (80) For 80 x 25 μl reactions* 204372

Rotor-Gene Probe PCR Kit (400) For 400 x 25 μl reactions* 204374

Rotor-Gene Probe RT-PCR Kit (400) For 400 x 25 μl reactions† 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

10 20 30 400.00

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B

QuantiFast Probe PCR Kit(IL8 transcript)

Kit from Supplier I(IL8 transcript)

1 ng cDNA

0.01 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 quantification from as little as 0.01 ng cDNA.

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

n Faster results with time savings of up to 60% n One procedure for all standard and fast cyclers n Sensitive detection of even low copy numbers n Accurate detection of a wide range of template amounts n 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, seepage 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 2–8°C. 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 Kit from Supplier I (IL12RBI transcript)

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 quantification from 0.1 ng RNA.

A QuantiFast Probe RT-PCR +ROX Vial Kit(IL12RBI transcript)

0.1 ng RNA

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.

L

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41%

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Two-step RT-PCR

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41%

40 60 80 100 120 1400

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One-step RT-PCR

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.











QuantiFast Probe PCR Kit (80) For 80 x 25 μl reactions* 204252

QuantiFast Probe PCR Kit (400) For 400 x 25 μl reactions* 204254

QuantiFast Probe PCR Kit (2000) For 2000 x 25 μl reactions*† 204256

QuantiFast Probe PCR +ROX Vial Kit (80) For 80 x 25 μl reactions‡ 204352

QuantiFast Probe PCR +ROX Vial Kit (400) For 400 x 25 μl reactions‡ 204354

QuantiFast Probe PCR +ROX Vial Kit (2000) For 2000 x 25 μl reactions†‡ 204356

QuantiFast Probe RT-PCR Kit (400) For 400 x 25 μl reactions§ 204454

QuantiFast Probe RT-PCR Kit (2000) For 2000 x 25 μl reactions†§ 204456

QuantiFast Probe RT-PCR +ROX Vial Kit (400) For 400 x 25 μl reactions¶ 204554

QuantiFast Probe RT-PCR +ROX Vial Kit (2000) For 2000 x 25 μl reactions†¶ 204556* 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




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 efficiency of 101%.

QuantiTect Probe Kits For real-time PCR and RT-PCR using sequence-specific probes

n Highly sensitive detection of low-copy targets n Accurate quantification over several logs of template n No need to optimize reaction and cycling conditions n Use of any sequence-specific probe on any real-time cycler n Available with or without uracil-N-glycosylase (UNG)

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 2–8°C. 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.









PCR efficiency: 94% PCR efficiency: 83%

CT = 34.64 CT = 36.92

QIAGENA Supplier AIIB

PCR efficiency: 94% PCR efficiency: 83%

CT = 34.64 CT = 36.92

QIAGENA Supplier AIIB

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 efficiency than the kit from Supplier AII.

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.




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


QuantiTect Probe PCR Kit (40) For 40 x 50 µl reactions* 204341

QuantiTect Probe PCR Kit (200) For 200 x 50 µl reactions* 204343

QuantiTect Probe PCR Kit (1000) For 1000 x 50 µl reactions*† 204345

QuantiTect Probe PCR +UNG Kit (200) For 200 x 50 µl reactions‡ 204363

QuantiTect Probe RT-PCR Kit (200) For 200 x 50 µl reactions§ 204443

QuantiTect Probe RT-PCR Kit (1000) For 1000 x 50 µl reactions†§ 204445* 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.




Improved detection of low amounts of viral RNA compared with other real-time kits. Viral RNA was diluted in serial fivefold dilutions and amplified 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.

QuantiTect Virus Kits For highly sensitive detection of viral RNA and/or DNA

n High sensitivity in singleplex and multiplex assays n Detection of viral RNA and/or DNA in the same reaction n Clear detection of weak positive signals n Fast universal 2-step protocols n 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.

ND

40

30

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Template dilution0.22.3 1.6 0.9 –0.5

C T




A12000

10000

8000

6000

4000

2000

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-2000

Unambiguous determination of CT values over a broad dynamic range. Serial dilutions of bovine viral diarrhea virus (BVDV) RNA, as indicated, were amplified 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/μl). A Detection of BVDV1 RNA. B Detection of BVDV2 RNA. NTC: No template control.

B80007000600050004000300020001000

0-1000

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

Related products



Automated purification and reaction setup QIAsymphony SP and AS 56

Viral nucleic acid purification QIAamp MinElute Virus Kits 279 QIAamp UltraSens Virus Kit 280 QIAamp Circulating Nucleic Acid Kit 278


QuantiTect Virus Kit (50) For 50 x 50 μl reactions* 211011



QuantiTect Virus +ROX Vial Kit (50) For 50 x 50 μl reactions† 211031



* 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.



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.

* Commercially available from other suppliers.

Type-it Fast SNP Probe PCR Kit For accurate and reliable SNP genotyping using TaqMan or TaqMan MGB™ probes

n Validated using TaqMan SNP Genotyping Assays* n Automated allele calling and tight fluorescence clusters n Suitable for difficult SNP loci or very low template amounts n 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.

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


Type-it Fast SNP Probe PCR Kit (100) For 100 x 25 μl reactions† 206042

Type-it Fast SNP Probe PCR Kit (800) For 800 x 25 μl reactions† 206045

Type-it Fast SNP Probe PCR Kit (4000) For 4000 x 25 μl reactions†‡ 206047† Includes Type-it Fast SNP Probe PCR Master Mix (with optimized MgCl2 concentration and 200 μM each dNTP), containing

HotStarTaq Plus DNA Polymerase, 5x Q-Solution, and RNase-free water. ‡ Master mix supplied in 2 individual tubes.




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

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 2–8°C. 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.

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 (β-actin). Analysis of tenfold dilutions of leukocyte cDNA template from 100 ng to 1 pg provided high PCR efficiencies of around 95%. C The CT values were comparable with those achieved in control singleplex reactions, demonstrating the reliability of the duplex assay.

CT values (IL8) CT values (ACTB)

Template Duplex Singleplex Duplex Singleplex

100 ng 15.17 15.70 14.78 14.25

10 ng 18.57 19.14 18.20 17.23

1 ng 21.51 22.65 21.77 21.26

100 pg 25.42 26.18 25.50 24.98

10 pg 28.96 29.41 29.01 28.48

1 pg 33.76 34.75 32.88 32.36

NTC 45.00 45.00 45.00 45.00

IL8 (FAM)

ACTB (HEX)

CT valuesC



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 amplified with the same high efficiency. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; RPS27A: ribosomal protein S27a; NFKB: nuclear factor of kappa light polypeptide gene enhancer in B-cells.


Rotor-Gene Multiplex PCR Kit (80) For 80 x 25 μl reactions* 204772

Rotor-Gene Multiplex PCR Kit (400) For 400 x 25 μl reactions* 204774

Rotor-Gene Multiplex RT-PCR Kit (80) For 80 x 25 μl reactions† 204972

Rotor-Gene Multiplex RT-PCR Kit (400) For 400 x 25 μl reactions† 204974* 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







C T

0

log pg template

35302520151050

GAPDH (HEX)RPS27A (CAL Fluor® Red 610)18S rRNA (Quasar® 670)

NFKB (Quasar 705)

1 2 3 4 5 6

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.




Kit from Supplier AII

(standard-cycling mode)B

103

108

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 β-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.

QuantiFast Multiplex Kits For fast multiplex real-time PCR and RT-PCR using sequence-specific probes

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 ■

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.


+ 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 2–8°C. 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.

108

10

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

A



A RPS27A (Cy5 dye) B GAPDH (HEX dye)

C 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 efficiencies (data not shown) demonstrate the reliability of triplex PCR with the QuantiFast Multiplex RT-PCR +R Kit when analyzing targets of differing abundance.

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.




Related products






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


QuantiFast Multiplex PCR Kit (80) For 80 x 25 μl reactions* 204652

QuantiFast Multiplex PCR Kit (400) For 400 x 25 μl reactions* 204654

QuantiFast Multiplex PCR +R Kit (80) For 80 x 25 μl reactions† 204752

QuantiFast Multiplex PCR +R Kit (400) For 400 x 25 μl reactions† 204754

New QuantiFast Multiplex RT-PCR Kit (80) For 80 x 25 μl reactions‡ 204852

New QuantiFast Multiplex RT-PCR Kit (400) For 400 x 25 μl reactions‡ 204854

New QuantiFast Multiplex RT-PCR +R Kit (80) For 80 x 25 μl reactions§ 204952

New QuantiFast Multiplex RT-PCR +R Kit (400) For 400 x 25 μl reactions§ 204954

* For use with all instruments from Applied Biosystems except the Applied Biosystems 7500. Includes 2x QuantiFast Multiplex PCR MasterMix (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-PCRMaster 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 2xQuantiFast Multiplex RT-PCR Master Mix (without ROX dye), 50x ROX Dye Solution, QuantiFast RT Mix, and RNase-free water.

16012080400

I 42%

48%

42%

41%

44%

39%

L1

L2

A1

A2

M

Time (min)

QuantiFast QuantiTect

Time savings

Two-step RT-PCRA

200150100500

I 38%

48%

45%

46%

41%

L1

L2

A2

M

Time (min)


Time savings

One-step RT-PCRBSignificantly 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.



Triplex PCRSingleplex PCR


Target 1 (FAM)

Target 2 (HEX)

Target 3 (Cy5)


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 Cy®5 dye were used. (Data kindly provided by the University of Minnesota, Minneapolis, MN, USA.)

QuantiTect Multiplex Kits For multiplex, real-time PCR and RT-PCR using sequence-specific probes

n Multiplex analysis with no need for optimization n Analysis of multiple targets in a single reaction n Sensitive detection of as few as 10 copies of each target n Reliable quantification of target and reference genes n 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.


+ 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 2–8°C. 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.




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 amplified and detected individually (black curves).

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)B

1000

Variable amounts of target RNA (FAM)A

1000

100

10

28S rRNA (triplex PCR)

POLD3 (triplex PCR)

CDK2 (triplex PCR)

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



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









QuantiTect Multiplex PCR Kit (40) For 40 x 50 μl reactions* 204541

QuantiTect Multiplex PCR Kit (200) For 200 x 50 μl reactions* 204543

QuantiTect Multiplex PCR Kit (1000) For 1000 x 50 μl reactions*† 204545

QuantiTect Multiplex PCR NoROX Kit (40) For 40 x 50 μl reactions‡ 204741

QuantiTect Multiplex PCR NoROX Kit (200) For 200 x 50 μl reactions‡ 204743

QuantiTect Multiplex PCR NoROX Kit (1000) For 1000 x 50 μl reactions†‡ 204745

QuantiTect Multiplex RT-PCR Kit (200) For 200 x 50 μl reactions§ 204643

QuantiTect Multiplex RT-PCR Kit (1000) For 1000 x 50 μl reactions†§ 204645

QuantiTect Multiplex RT-PCR NR Kit (200) For 200 x 50 μl reactions¶ 204843

QuantiTect Multiplex RT-PCR NR Kit (1000) For 1000 x 50 μl reactions†¶ 204845* 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.




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.





Type-it HRM PCR Kit (100)* For 100 reactions 206542

Type-it HRM PCR Kit (400)* For 400 reactions† 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

5

0

-15

-10

-5

2x HRM Master Mix (Q); 25 µl/reaction

76.5

77.0

77.5

78.0

78.5

79.0

79.5

80.0

80.5

81.0

Clear separation from wild type

A

5

0

-15

-10

-5

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

77.5

78.0

78.5

79.0

79.5

80.0

80.5

81.0

Incorrect genotyping

82.5

82.0

81.5

B

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 confidence 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.

Related productsReal-time cycler Rotor-Gene Q 78






Highly specific cloning with a short 30-minute ligation time. The effect of ligation time on cloning efficiency 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).

QIAGEN PCR Cloningplus Kit For direct cloning of PCR products

n Just 40 minutes from PCR product to plated cells n Ready-to-use Ligation Master Mix n Immediate plating of transformed competent cells n High-specificity UA hybridization for efficient cloning n 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.

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

0

20

40

60

80

100

120

0.5 kb 3 kb1 kb


QIAGEN PCR Cloningplus Kit (10) For 10 reactions* 231222

QIAGEN PCR Cloningplus Kit (40) For 40 reactions* 231224* Includes 2x ligation master mix, pDrive cloning vector, distilled water, QIAGEN EZ competent cells, and SOC medium.



Cloning 10.3

Robust QIAGEN EZ Competent Cells do not require recovery incubation. QIAGEN EZ Competent Cells (>108 cfu/μg DNA), TOP 10F (Supplier I; >109 cfu/μg DNA), and JM109 (Supplier P;>108 cfu/μg 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 efficiency. Normalization would result in an even higher transformation efficiency for QIAGEN EZ Competent Cells. (QIAGEN EZ Competent Cells are supplied with the QIAGEN PCR Cloningplus Kit.)

QIAGEN PCR Cloning Kit For direct cloning of PCR products generated by Taq DNA polymerases

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.

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

0

20

40

60

80

100

120

QIAGEN

EZ

Compe

tent C

ells

JM10

9

TOP 1

0F


QIAGEN PCR Cloning Kit (10) For 10 reactions* 231122

QIAGEN PCR Cloning Kit (40) For 40 reactions* 231124

* Includes 2x ligation master mix, pDrive cloning vector, and distilled water.

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