amplification pcr reagents - chemistry

PCR Reagents

The Highest Level of PCR Performance

· PCR Enzymes· RT-PCR Reagents· QPCR and QRT-PCR Reagents· dNTPs

A m p l i f i c a t i o n

Stratagene PCR Enzymes www.stratagene.com

Stratagene Has Your PCR ReagentHigh-Fidelity Applications

GeneralCloning

TA/UACloning

LongTargets

PfuUltra™

High-FidelityDNA Polymerases

Easy-A™ High-Fidelity

PCR Cloning Enzyme

EXL™

DNA Polymerase

PfuTurbo®

DNA Polymerases

PfuTurbo PfuTurbo® Cx

Hotstart DNA Polymerase

RECOMMENDED

GOOD

KITS & MASTER MIXES

Herculase®

EnhancedDNA Polymerases

PfuUltra™

PCR Master Mix PfuTurbo®

PCR Master MixEasy-A™

PCR Master Mix

PfuUltra page 6

page 7

page 8

PfuTurbo PfuTurbo page9

page 10

Herculase page 10

page 6

PfuTurbo page 8 page 7

Stratagene PCR Enzymes www.stratagene.com

Stratagene Has Your PCR ReagentHigh-Fidelity Applications General Applications

LongTargets

Complex/GC-RichTargets

Multiple Targets Routine PCR QPCR/

Real TimeReverse

Transcription

www.stratagene.com

The ArchaeMaxx™ Factor Advantage• Higher yields• Shorter extension times• Greater target length capability

A key component found exclusively in many of Stratagene’s high-fi delity Pfu-based DNA polymerases is the patented ArchaeMaxx™ polymerase-enhancing factor***, which improves overall PCR performance.

The ArchaeMaxx factor, discovered by Stratagene scientists, eliminates the inhibition of proofreading enzymes caused by incorporation of dUTP, which results from deamination of dCTP during PCR1.

EXL™

DNA Polymerase

Herculase®


StrataScript™

ReverseTranscriptase

SureStart®

Taq DNAPolymerase

TaqPlus® Maxx™

DNAPolymerase

StrataScript™

RT-PCRSystems

Brilliant®

QPCRReagent Kits

Taq2000™

DNA Polymerase

Herculase®


SureStart®

Taq DNAPolymerase

Herculase®


Herculase®

PCR Master Mix

PfuTurbo PfuTurbo® Cx

Hotstart DNA Polymerase

Herculase page 10

TaqPlus page 11

Herculase page 10

page 12

SureStart page 12

StrataScript page 13

SureStart page 12

Brilliant page 14

StrataScript page 13

PfuTurbo PfuTurbo page 9

Herculase page 10

P C R R E AG E N T S w w w. s t ra t a g e n e . c o m4 5

Expertise Breeds InnovationOur expertise in enzyme engineering keeps us on the leading edge of PCR enzyme development. As the leader in high-fidelity PCR, we offer one of the broadest portfolios of high-fidelity PCR enzymes available today. Whether you need the highest accuracy available, large product yields, the ability to amplify long or GC-rich targets, or the most reliable enzyme no matter how many targets you have, trust our high-performance polymerases to answer your PCR challenges.

High Performance Means High Amplification EfficiencyAmplification efficiency is a measure of the percentage of PCR product that doubles with each cycle. Many factors can affect amplification efficiency, including the performance of the polymerase. A slight change in efficiency, magnified over 30 cycles, can have a significant effect on product yield. Quantitative or real-time PCR provides a true determination of amplification efficiency because it can measure results before saturation is reached.

Recent testing has shown that our Pfu-based enzymes containing the ArchaeMaxx™ polymerase-enhancing factor exhibit higher amplification efficiencies than other commercially available enzymes2.

Stratagene – The Leader in High-Performance PCR

Pfu-Based Enzymes Exhibit Higher EfficienciesTemplates of 0.9 kb, 2.6 kb, and 3.9 kb with similar GC-content (53 to 56%) were amplified using various commercially available PCR enzymes. Efficiencies shown are averages of at least three independent experiments. Efficiencies of systems using Platinum Pfx DNA polymerase at 2.6 kb and 3.9 kb and Taq DNA polymerase at 3.9 kb could not be detected due to the presence of multiple non-specific products at high concentrations and no measurable product at lower concentrations.

0.9 kb 2.6 kb 3.9 kbND*

10

20

30

40

50

60

70

80

90

*ND= Not Detected

Effic

ienc

y

PfuUltra®, PfuTurbo®, and Herculase® DNA Polymerases

Pfu DNA Polymerase

Taq DNA Polymerase

Platinum® Pfx DNA Polymerase (Invitrogen)


The Importance of High FidelityHigh-fidelity PCR enzymes are valuable for minimizing the introduction of amplification errors in products that will be cloned, sequenced, and expressed. Significant time and effort can be saved by employing high-fidelity amplification procedures that eliminate the need for downstream error-correction steps and minimize the number of clones that must be sequenced in order to obtain error-free constructs or accurate consensus sequences. Moreover, the use of high-fidelity amplification conditions is essential when analyzing very small amounts of template DNA or rare molecules in heterogeneous populations. Amplifications employing small amounts of template DNA are especially prone to high mutant frequencies, due to PCR-generated errors in early cycles (“jackpot” artifacts) and high target doublings3,4.

Is Sequence Verification Breaking Your Bank?Sequence verification is an expensive portion of the total cost of a PCR cloning project. When you use one of our high-fidelity PCR enzymes, you can sequence fewer clones with high confidence that you will obtain your desired product error-free. The chart below illustrates the probability of an error-free product using various commercially available PCR enzymes based on amplicon length and the intrinsic error-rate of each enzyme determined using our published PCR fidelity assay5.

* Mutation Freq. = (Enzyme Error Rate)*(# bp)*(# doublings) and assumes 106-fold amplification (# doublings = 20)‡ Calculated using the formula: p = 1 – (1-f)n where p = probability, f = frequency of obtaining the correct clone, and n = number of clones

sequenced

Green shaded areas indicate ≥99.0% probability of obtaining a correct clone based on the number of clones sequenced. Red shaded areas indicate ≥99.0% probability of obtaining a correct clone; increased chance that all sequenced clones will contain errors.

4.2 kb amplicon Blunt Mutation % Probability‡ of a Correct Clone if You… or 3'-A Frequency Sequence Sequence Sequence Sequence SequencePCR Enzyme Ends (%)* 1 Clone 2 Clones 3 Clones 4 Clones 12 Clones

PfuUltra™ Hotstart DNA Polymerase Blunt 3.4 96.60 99.88 PfuTurbo® and PfuTurbo® Cx DNA Polymerases Blunt 10.9 89.10 98.80 99.87 Easy-A™ High-Fidelity PCR Cloning Enzyme 3'-A 10.9 89.10 98.80 99.87 Vent® DNA Polymerase Blunt 23.5 76.50 94.50 98.70 99.70 Platinum® Pfx DNA Polymerase Blunt 29.0 71.00 91.60 97.60 99.30 Taq DNA Polymerase 3'-A 67.0 33.00 55.00 70.00 80.00 99.20

1.2 kb amplicon Blunt Mutation % Probability‡ of a Correct Clone if You… or 3'-A Frequency Sequence Sequence SequencePCR Enzyme Ends (%)* 1 Clone 2 Clones 3 Clones

PfuUltra™ Hotstart DNA Polymerase Blunt 1.0 99.00 99.99 PfuTurbo® and PfuTurbo® Cx DNA Polymerases Blunt 3.1 96.90 99.90 Easy-A™ High-Fidelity PCR Cloning Enzyme 3'-A 3.1 96.90 99.90 Vent® DNA Polymerase Blunt 6.7 93.30 99.55 Platinum® Pfx DNA Polymerase Blunt 8.4 91.60 99.29

Taq DNA Polymerase 3'-A 19.2 80.80 96.30 99.29


PfuUltra™ high-fidelity DNA polymerase*,‡ is an enzyme formulation containing a genetically engineered mutant of Pfu DNA polymerase** that exhibits enhanced proofreading capability. The accuracy of PfuUltra DNA polymerase was compared to Pfu and Taq DNA polymerases using a validated and referenced fidelity assay5. Our data demonstrates that PfuUltra high-fidelity DNA polymerase exhibits an average error rate three-fold lower than Pfu DNA polymerase and 18-fold lower than Taq DNA polymerase, making it the most accurate enzyme available. The addition of the ArchaeMaxx™ polymerase-enhancing factor*** promotes higher yields, shorter extension times and greater target length capability.

ALSO AVAILABLE:

PfuUltra ™ Hotstart DNA Polymerase Antibody-based hot-start formulation

PfuUltra ™ Hotstart PCR Master Mix 2X formulation including polymerase, buffer, dNTPs and MgCl2

Highest Accuracy

• The most accurate PCR enzyme available

• Pfu mutant delivers 300% greater accuracy

• Ideal for PCR cloning and site-directed mutagenesis

Delivers the Greatest Accuracy of Any Available EnzymePfuUltra™ high-fidelity DNA polymerase provides the greatest accuracy of any commercially available enzyme. Fidelity was measured using Stratagene’s validated and referenced fidelity assay. Accuracy = 1/Error Rate.

PfuUltra™ DNA Polymerases

PfuUltra™ High-Fidelity DNA Polymerase easily amplifies genomic DNA targets up to 17 kb.

Kb M 0.97 1.5 3.5 6 9 12 17

PfuTurbo® and PfuTurbo® Cx

DNA Polymerases

PfuUltra™ DNAPolymerase

Pfx/KOD DNAPolymerases

Herculase® DNAPolymerase

Expand™ HighFidelity

Advantage-HF™

PolymeraseTgo DNA

PolymeraseTaq DNA

Polymerase

Accuracy

Acc

ura

cy x

105

DNA Polymerases

25

20

15

10

5

0

Length 0-17 kb (genomic) 0-15 kb (vector)Extension time 1 min/kb (0-6 kb) 2 min/kb (>6 kb)Blunt or 3'-A ends BluntAccuracy vs. Taq 18XArchaeMaxx™ factor advantage

Hot-start versionavailable

Master mix formatavailable


Easy-A™ high-fidelity PCR cloning enzyme*,‡ is the only proofreading DNA polymerase formulation to offer high-throughput cloning into T- or /U-modified vectors. PCR products amplified with the Easy-A cloning enzyme can be cloned directly, without performing additional steps typically required when amplifying with proofreading polymerases.

Adapting blunt-ended fragments amplified with proofreading enzymes requires post-PCR addition of 3'-A overhangs prior to the cloning step. These additional enzymatic steps include a secondary incubation with Taq DNA polymerase and require the opening and closing of tubes, which is detrimental to sample throughput and exposes the lab to contamination risk. Easy-A high-fidelity PCR cloning enzyme does not require any post-PCR A-addition steps. Thus, PCR products generated with the Easy-A PCR cloning enzyme can be added directly to T- or U-modified vectors to provide improved TA/UA cloning without sacrificing accuracy, cloning efficiency, or throughput.

ALSO AVAILABLE:

Easy-A™ High-Fidelity PCR Master Mix 2X formulation including polymerase, buffer, dNTPs and MgCl2

Efficient, Accurate TA/UA Cloning

High Cloning Efficiency with Easy-A™ PCR Cloning EnzymeTargets amplified with Easy-A™ PCR Cloning enzyme contain 3'-A overhangs, allowing quick and easy cloning into any T- or U-modified vector.

Easy-A™ High-Fidelity PCR Cloning Enzyme • Combines the cloning efficiency of Taq DNA polymerase with the accuracy of Pfu DNA polymerase

• Proofreading DNA polymerase formulation adds 3'-A overhangs to PCR amplicons as efficiently as Taq

• High throughput method eliminates post-PCR A-addition steps and contamination risk

• Hot-start formulation provides enhanced specificity

T-vector

Ligation complete

Add 0.5-1.5 µl of PCR productto T-modified vector

Topoisomerase-mediated ligation or conventional ligation

Ligation

Ligation complete

T-vector U-vector U-vector

5' 3'

3'

5'

3'

5'

3'

5'

5'

3'

3'

5'

5' 3'

3' 5'

PCR product

TT

UU

UUAAT

TAA

AA

AA

Add 0.5-1.5 µl of PCR productto U-modified vector

Easy-A™ -amplifiedPCR products

Easy-A™-amplifiedPCR products

PCR product

T-vector

T-vectorU-vector U-vector

Length 0-5 kb Extension time 1 min/kbBlunt or 3'-A ends 3'-AAccuracy vs. Taq 6XArchaeMaxx™ factor advantage

Hot-start version Master mix formatavailable

P C R R E AG E N T S w w w. s t ra t a g e n e . c o m

PfuTurbo ® DNA Polymerase Generates High Yields of Accurate PCR ProductsPfuTurbo ® DNA polymerase was used to amplify portions of the human α-1 antitrypsin gene. PCR product was easily generated up to 19 kb.

8 9

High-Fidelity

PfuTurbo® DNA Polymerases• Same high fidelity as Pfu DNA polymerase with superior performance

• Requires shorter extension times, fewer PCR cycles and less DNA

• Ideal for amplifying complex genomic DNA targets

PfuTurbo® DNA polymerase*,‡ is a special formulation of cloned Pfu DNA polymerase** and the novel ArchaeMaxx™ polymerase-enhancing factor*** that significantly increases PCR product yields without affecting replication fidelity. The enhanced performance of PfuTurbo DNA polymerase allows the use of shorter extension times, fewer PCR cycles and lower concentrations of DNA template than are required for Pfu DNA polymerase.

ALSO AVAILABLE:

PfuTurbo ® Hotstart DNA Polymerase Antibody-based hot-start formulation

PfuTurbo ® Hotstart PCR Master Mix 2X formulation including polymerase, buffer, dNTPs and MgCl2

0.9 2.1 4 6 9.3 11.9 17 19 M

Pfu DNA PolymerasePfu DNA polymerase*,‡, derived from the hyperthermophilic archae Pyrococcus furiosus, has been shown to exhibit superior thermostability and proofreading properties compared to other thermostable polymerases.5 Unlike Taq DNA polymerase, highly thermostable Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity that enables the polymerase to correct nucleotide-misincorporation errors. Cloned Pfu DNA polymerase is a recombinant version of native Pfu that provides superior consistency and minimal lot-to-lot variability.

Length 0-19 kb (genomic) 0-15 kb (vector)Extension time 1 min/kb (0-6 kb) 2 min/kb (>6 kb)Blunt or 3'-A ends BluntAccuracy vs. Taq 6XArchaeMaxx™ factor advantage



Length 0-4 kbExtension time 2 min/kbBlunt or 3'-A ends BluntAccuracy vs. Taq 6XArchaeMaxx™ factor advantage



P C R R E AG E N T S w w w. s t ra t a g e n e . c o m

PfuTurbo Cx Hotstart DNA Polymerase Successfully Incorporates dUTPA 900-bp α-1 anti-trypsin genomic DNA target was amplified with PfuTurbo Cx hotstart DNA polymerase and original PfuTurbo hotstart DNA polymerase using nucleotide mixes including 200µM dTTP or 200µM dUTP. Analysis was performed on Stratagene’s Mx3000P™ real-time PCR system. Data shows no amplification with PfuTurbo DNA polymerase using dUTP, but nearly identical amplification results from PfuTurbo Cx hotstart DNA polymerase with either dTTP or dUTP.

PfuTurbo® Hotstart,200 µM dTTP

PfuTurbo® Cx Hotstart,200 µM dUTP

PfuTurbo® Cx Hotstart,200 µM dTTP

PfuTurbo® Hotstart,200 µM dUTP

PfuTurbo® Cx AccuPrime™ Pfx Platinum® Pfx

9 kb

6 kb

2.6 kb

0.9 kb

Superior Performance with PfuTurbo® Cx Hotstart DNA PolymeraseAmplification of a range of targets from 0.9 to 9 kb using PfuTurbo® Cx DNA polymerase and other commercially available proofreading PCR enzymes.

8 9

• Reliable, accurate amplification

• Tolerates variations in PCR conditions

• Fidelity equivalent to Pfu and PfuTurbo® DNA polymerases

• Incorporates dUTP for use with UNG decontamination protocol

...and High Performance

PfuTurbo ® Cx Hotstart DNA PolymerasePfuTurbo® Cx hotstart DNA polymerase*,‡ combines high fidelity with trouble-free PCR performance. Formulated with a new mutant of Pfu DNA polymerase, PfuTurbo Cx DNA polymerase easily amplifies problematic PCR templates with high accuracy, surpassing other proofreading PCR enzymes in overall performance, and making it the most versatile and reliable proofreading enzyme available.

Unlike other proofreading PCR enzymes which stop replication just before reaching a uracil residue6, the novel Pfu mutant in PfuTurbo Cx DNA polymerase allows the enzyme to read through a uracil located in the template strand without stalling. Thus PfuTurbo Cx DNA polymerase improves the overall reliability of high-fidelity PCR and exhibits less finicky, more robust performance. This product is ideal for amplification of templates of 5 to 10 kb in length, as well as from more difficult systems including targets with high GC content.

Although most systems are amplified successfully without the need for additives, DMSO is included with PfuTurbo Cx hotstart DNA polymerase to further enhance yields of longer or especially problematic sequences.

Length 0-10 kb Extension time 1 min/kb (0-6 kb) 2 min/kb (>6 kb)Blunt or 3'-A ends BluntAccuracy vs. Taq 6XArchaeMaxx™ factor advantage



Difficult & GC-Rich Templates / Extra-Long Targets

Herculase +/- DMSO (%)Competitor’s GC-rich PCR Enzyme

+/- GC-rich solution (M)0 06 7 8 9 0.5 1.0 1.5 2.0

1 2 3 4 5 6 7 8 9 10M

• Successfully amplifies targets >20 kb

• Higher fidelity than Taq and Taq-based enzyme blends

• Effectively amplifies problematic templates, including complex and GC-rich

• Amplifies a broad range of target lengths

• Higher fidelity than Taq and Taq-based enzyme blends

Herculase® enhanced DNA polymerase*,‡ meets today’s complex PCR challenges. When you need to accurately amplify a complex or GC-rich template, Herculase DNA polymerase will help you succeed. A unique optimized formulation of high-fidelity Pfu DNA polymerase**, Taq DNA polymerase, and the ArchaeMaxx™ polymerase-enhancing factor***, Herculase DNA polymerase excels in amplifying a broad range of target lengths with a more robust yield and superior fidelity than Taq DNA polymerase and other Taq-based blends7. DMSO is provided separately as a PCR adjunct, and can be added when amplifying difficult targets to increase product yield and extend target-length capability.

ALSO AVAILABLE:

Herculase ® Hotstart DNA Polymerase Antibody-based hot-start formulation

Herculase ® Hotstart PCR Master Mix 2X formulation including polymerase, buffer, dNTPs and MgCl2

Herculase Enhanced DNA Polymerase Excels in Amplifying Difficult TargetsHerculase enhanced DNA polymerase easily amplifies an 82.5% GC-rich fragment of Fragile X gene from human genomic DNA.

EXL™ DNA PolymeraseEXL™ DNA polymerase*,‡ provides superior performance in amplifying complex targets greater than 20 kb in length, with higher fidelity than Taq DNA polymerase or Taq-based blends. EXL DNA polymerase is a special formulation of Pfu DNA polymerase**, Taq DNA polymerase, and the ArchaeMaxx™ polymerase enhancing factor***, optimized specifically for extremely long targets and providing robust yield, specificity, and reliable amplification.

M 23 2330 3045 45EXL Competitor

EXL™ DNA Polymerase Excels at Amplifying Extremely Long TargetsEXL DNA polymerase easily amplifies two extremely long genomic targets (23 and 30 kb)and a lambda target (45 kb). A competitor’s PCR enzyme for long targets was used for comparison. All reactions were performed according to the manufacturer’s recommendations.

Herculase® DNA Polymerases

Length 0-37 kb (genomic) 0-48 kb (vector)Extension time 1 min/kbBlunt or 3'-A ends MixedAccuracy vs. Taq 3.5XArchaeMaxx™ factor advantage



Length 20-37 kb (genomic) 20-50 kb (vector)Extension time 1 min/kbBlunt or 3'-A ends MixedAccuracy vs. Taq 3.5XArchaeMaxx™ factor advantage




Highest PCR Success Rate

• Highest success rate of any PCR polymerase or enzyme blend

• Consistently high yields on a variety of templates up to 10 kb

• Greater sensitivity allows successful amplification where starting material is limited

• High-specificity hot-start allows reliable room-temperature setup

TaqPlus ® Maxx™ DNA PolymeraseTaqPlus® Maxx™ DNA polymerase*,‡ is engineered for maximum PCR reliability. TaqPlus Maxx DNA polymerase is a blend of cloned Taq and Pfu DNA polymerases, and the ArchaeMaxx™ polymerase-enhancing factor***. Together with a specially optimized buffer, this enzyme blend provides the highest success rate of any PCR enzyme, including other Taq-based blends. TaqPlus Maxx DNA polymerase reliably produces high PCR product yields on a wide variety of targets up to 10 kb in length.

Amplification from small quantities of template can be difficult for most PCR enzymes. TaqPlus Maxx DNA polymerase provides superior PCR sensitivity and can therefore amplify successfully from a fraction of the template quantity required by other polymerases. High sensitivity permits preservation of samples where starting material is limited, and allows TaqPlus Maxx DNA polymerase to detect samples that might otherwise be missed, further adding to its reliability.

TaqPlus® Maxx™ DNA Polymerase Exhibits Superior SensitivityA 3.9 kb α-1 antitrypsin template was amplified with TaqPlus® Maxx™ DNA polymerase, Expand™ High Fidelity PCR System and Platinum® Taq DNA Polymerase High Fidelity using decreasing template quantities.

TaqPlus® Maxx™ DNA Polymerase Provides Reliable PCR ResultsTemplates ranging from 0.9 kb to 9 kb were amplified using TaqPlus® Maxx™ DNA polymerase, Expand™ High Fidelity PCR System and Platinum® Taq DNA Polymerase High Fidelity. All reactions were performed according to the manufacturer’s recommendations.

50 25 10 1 0 50 25 10 1 0 50 25 10 1 0

3.9 kb

TemplateQty. (ng)

PCR Enzyme Blends

TaqPlus® Maxx™ Expand™ High Fidelity Platinum® Taq High FidelityDeoxynucleotide Mix

• Produces high-quality reaction products

• Withstands multiple freeze-thaw cycles without compromising efficiency

Our dNTP mix contains 400 µl of 100 mM dNTP mix (25 mM of each dNTP), adequate for 500 -1000 standard 100 µl primer-extension reactions.

9 kb

4 kb

1.7 kb

0.9 kb

TaqPlus® Maxx™Expand™

High-FidelityPlatinum® Taq High-Fidelity

PCR Enzyme Blends

Length 0-10 kb Extension time 1 min/kbBlunt or 3'-A ends MixedAccuracy vs. Taq 2XArchaeMaxx™ factor advantage



Routine PCR

• Hot-start formulation of Taq DNA polymerase

• Provides greater PCR specificity and yields

• Reduces nonspecific background

• Allows reliable room-temperature setup

SureStart® Taq DNA PolymeraseSureStart® Taq DNA polymerase‡ is a hot-start Taq DNA polymerase. SureStart Taq can be incorporated into PCR protocols previously optimized with Taq DNA polymerase, with little or no modification of cycling parameters or reaction conditions. This specially modified Taq DNA polymerase allows setup of PCR reactions at ambient room temperature without nonspecific annealing and extension during PCR setup, making setup easier. SureStart Taq DNA polymerase can be used in a variety of amplification systems to improve specificity, yield, and amplification of difficult targets.

Taq2000 ™ DNA Polymerase• Ultra-pure cloned Taq DNA polymerase

• Minimizes smearing• Highly thermostable Taq2000™ DNA polymerase‡ is a highly purified, recombinant Taq DNA

polymerase cloned from the thermophilic eubacteria, Thermus aquaticus. Using Taq2000 DNA polymerase in longer PCR amplifications reduces smearing and virtually eliminates unwanted background artifacts. Taq2000 DNA polymerase has superior thermostability compared to other commercial Taq DNA polymerase preparations.

Length 0-4 kbExtension time 1 min/kbBlunt or 3'-A ends 3'-AAccuracy vs. Taq 1XArchaeMaxx™ factor advantage



SureStart® Taq DNA Polymerase Increases Specificity A 105 bp fragment of the glucocerebrosidase gene was amplified from human genomic DNA. Reactions were assembled at room temperature and employed 2.5 U of each enzyme and the recommended buffer and cycling parameters. Lane 1: SureStart Taq DNA polymerase, lane 2: unmodified Taq DNA polymerase, lane 3: an antibody-based hot start Taq DNA polymerase, lane 4: competitor’s modified Taq DNA polymerase.

Taq2000™ DNA Polymerase vs. Competitor’s Cloned Taq DNA PolymerasePCR smear artifacts were produced with increasing extension times noted. A 4-kb fragment of transgenic mouse genomic DNA was amplified using Stratagene’s Taq2000 DNA polymerase versus another major manufacturer’s cloned Taq DNA polymerase.

Length 0-5 kbExtension time 1 min/kbBlunt or 3'-A ends 3'-AAccuracy vs. Taq 1XArchaeMaxx™ factor advantage




RT-PCR

• RNase H deficiency generates longer transcripts

• Nuclease-free enzyme produces robust cDNA yields

• Ideal enzyme for cDNA library construction and RT-PCR

StrataScript™ Reverse Transcriptase

• Generates high-quality cDNA templates from total or poly(A)+ RNA isolated from a variety of cells and tissue

• Applies the first-strand synthesis technique for PCR amplification of specific cDNA fragments

• Generates a heterogeneous population of cDNA molecules

StrataScript™ first strand cDNA synthesis kit employs StrataScript RNase H(-) reverse transcriptase to generate cDNA of high quality. Subsequent amplification with sequence-specific primers yields a homogenous population of the specific cDNA molecule of interest, eliminating the need for amplification and screening of a library of cDNA molecules.

• High sensitivity achieved from a wide variety of RNAs

• Ideally suited for cloning, expression, and sequencing

• One-tube kit allows quick, sensitive, and reproducible analysis of RNA with convenience and minimal risk of sample contamination

• Two-tube kit allows archive of cDNA samples before amplification step

StrataScript™ one-tube RT-PCR system includes Easy-A™ PCR cloning enzyme, a high-fidelity DNA polymerase with terminal transferase activity. Amplified products will contain fewer errors and are ready for subsequent TA/UA cloning with no additional steps required.

StrataScript™ two-tube RT-PCR system includes PfuUltra™ high-fidelity DNA polymerase, the most accurate PCR enzyme available. Both kits contain StrataScript reverse transcriptase.

Same Robust Performance Reactions using 100 U StrataScript™ reverse transcriptase and 100 U SuperScript™ II reverse transcriptase generated equivalent performance at 40 ˚C.

StrataScript™ reverse transcriptase is a Moloney murine leukemia virus reverse transcriptase (MMLV RT) that has been genetically modified to remove RNase H activity. The mutation in the highly conserved residue of the RNase H region results in the loss of RNase H activity without affecting the desired reverse transcriptase function. As a result, StrataScript RT generates longer transcripts and is the preferred enzyme for cDNA synthesis. This nuclease-free MMLV RT yields much larger quantities of full-length cDNA transcripts than wild-type MMLV RT, which possesses substantial RNase H activity.

StrataScript™ First Strand cDNA Synthesis Kit

StrataScript™ RT-PCR Systems

100 UStrataScript™

RT

100 USuperScript™ II

RT


QPCR and QRT-PCR

• Optimized on our Mx4000® and Mx3000P™ real-time PCR systems

• Master mixes offer convenience and maximum throughput

• Core reagent kits allow optimization of reactions

• SureStart® Taq DNA polymerase provides hot-start specificity

• StrataScript™ RNase H(-) reverse transcriptase provides superior sensitivity

Brilliant® QPCR and QRT-PCR reagents provide maximum versatility in real-time PCR detection. All Brilliant reagent kits contain SureStart® Taq DNA polymerase, a hot-start version of Taq that minimizes amplification of non-specific PCR products. Brilliant reagent kits are optimized for use on our Mx4000® and Mx3000P™ real-time PCR systems, but can be used on virtually any QPCR instrument. Whether you choose to optimize your reactions with a core reagent kit or prefer the convenience of a master mix, there is a Brilliant QPCR or QRT-PCR reagent kit for you.

Brilliant® probe-based core reagent kits and master mixes are compatible with any fluorescent probe chemistry including TaqMan® probes, Molecular Beacons, and Scorpions™. Our probe-based core kits are available with a standard nucleotide mix or with a GAUC mix (“Plus” kits) for dUTP/UNG decontamination strategy.

Brilliant® SYBR® Green QPCR reagents provide a universal solution to real-time PCR detection and gene quantification, without the need for sequence-specific probes. Choose Brilliant SYBR Green master mixes for maximum convenience, or optimize your reactions using the SYBR Green core reagent kit.

Select the Right Brilliant® Reagent for You

SYBR® GreenMaster Mix

Probe-BasedMaster Mix

SYBR® GreenCore Kit

Probe-Based Core Kits

(GAUC in Plus kit)

Master Mixes Core Reagent Kits

QPCR (DNA)

SYBR® GreenMaster Mix

Probe-BasedMaster Mix

Probe-Based 2-Step Kits

(GAUC in Plus kit)

Master Mixes

QRT-PCR (RNA)

Probe-Based 1-Step Kits

(GAUC in Plus kit)

Core Reagent Kits

Brilliant® QPCR Master Mixes & Core Reagent Kits


Ordering Information

Brilliant® QPCR Master Mixes & Core Reagent Kits PAGE 14 continued

PROBE-BASED CORE REAGENT KITS

Brilliant® QPCR Core Reagent Kit200 rxn 6005302000 rxn 929530

Brilliant® QPCR Plus Core Reagent Kit (dUTP/UNG)200 rxn 6005402000 rxn 929540

Brilliant® QRT-PCR Core Reagent Kit, 1-Step200 rxn 6005322000 rxn 929532

Brilliant® QRT-PCR Core Reagent Kit, 2-Step200 rxn 6005342000 rxn 929534

Brilliant® QRT-PCR Plus Core Reagent Kit, 1-Step (dUTP)200 rxn 6005422000 rxn 929542

PAGE 10 continuedHerculase® Enhanced DNA Polymerase100U 600260500U 6002621000U 600264

Herculase® Hotstart DNA Polymerase100U 600310500U 6003121000U 600314

Herculase® Hotstart PCR Master Mix100 rxn 600610400 rxn 600612

PAGE 11TaqPlus® Maxx™ DNA Polymerase100U 600420500U 6004221000U 600424

PAGE 12SureStart® Taq DNA Polymerase100U 600280500U 6002821000U 600284

Taq2000™ DNA Polymerase100U 600195500U 6001961000U 600197

PAGE 13StrataScript™ First Strand RT-PCR Kit50 rxn 200420

StrataScript™ One-Tube RT-PCR System50 rxn 600168

StrataScript™ Reverse Transcriptase10,000U 600085

StrataScript™ Two-Tube RT-PCR System50 rxn 600170

PAGE 14Deoxynucleotide Mix400 µl 200415

PROBE-BASED MASTER MIXES

Brilliant® QPCR Master Mix200 rxn 6005492000 rxn 929549

Brilliant® QRT-PCR Master Mix Kit, 1-Step200 rxn 6005512000 rxn 929551

PAGE 6PfuUltra™ High-Fidelity DNA Polymerase100U 600380500U 6003821000U 600384

PfuUltra™ Hotstart DNA Polymerase100U 600390500U 6003921000U 600394

PfuUltra™ Hotstart PCR Master Mix100 rxn 600630400 rxn 600632

PAGE 7Easy-A™ High-Fidelity PCR Cloning Enzyme100U 600400500U 6004021000U 600404

Easy-A™ High-Fidelity PCR Master Mix100 rxn 600640400 rxn 600642

PAGE 8Cloned Pfu DNA Polymerase100U 600153500U 6001541000U 600159

Native Pfu DNA Polymerase100U 600135500U 6001361000U 600140

PfuTurbo® DNA Polymerase100U 600250500U 6002521000U 600254

PfuTurbo® Hotstart DNA Polymerase100U 600320500U 6003221000U 600324

PfuTurbo® Hotstart PCR Master Mix100 rxn 600600400 rxn 600602

PAGE 9PfuTurbo® Cx Hotstart DNA Polymerase100U 600410500U 6004121000U 600414

PAGE 10EXL™ DNA Polymerase100U 600340500U 6003421000U 600344

PAGE 14 continuedBrilliant® QRT-PCR Plus Core Reagent Kit, 2-Step (dUTP/UNG)200 rxn 6005442000 rxn 929544

SYBR® GREEN MASTER MIXES

Brilliant® SYBR® Green QPCR Master Mix200 rxn 6005482000 rxn 929548

Brilliant® SYBR® Green QRT-PCR Master Mix Kit, 1-Step200 rxn 6005522000 rxn 929552

SYBR® GREEN CORE REAGENT KITS

Brilliant® SYBR® Green QPCR Core Reagent Kit200 rxn 6005462000 rxn 929546

Legal Information

Scorpions™ is a trademark of DxS Ltd.

SYBR® is a registered trademark of Molecular Probes.

TA Cloning® is a registered trademark of Invitrogen Corp.

TaqMan® is a registered trademark of Roche Molecular Systems, Inc.

*U.S. Patent Nos. 6,444,428, 6,379,553, 6,333,165, 6,183,997, 6,489,150, 5,948,663, 5,866,395, 5,556,772, 5,545,552 and patents pending.**U.S. Patent Nos. 6,489,150, 5,948,663, 5,866,395, 5,545,552 and patents pending.***U.S. Patent Nos. 6,444,428, 6,379,553, 6,333,165, 6,183,997 and patents pending.

‡Purchase of these products is accompanied by a license to use them in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Applied Biosystems or as purchased, i.e., an authorized thermal cycler.

References1. Hogrefe, et al. (2002). Proc. Nat. Acad. Sci. USA 99: 596-601.2. Strategies 16.3, p. 96-97.3. Hogrefe, H.H. and M.C. Borns. High Fidelity PCR Enzymes. In C.W.

Dieffenbach, G.S. Dveksler (eds.) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003.

4. Cha, R.S. and W.G. Thilly. Specificity, efficiency, and fidelity of PCR. In PCR Primer: A Laboratory Manual (eds, Dieffenbach, C.W. and G.S. Dveksler) Cold Spring Harbor Laboratory Press, 1995.

5. Cline, J., Braman, J.C. and Hogrefe, H.H. (1996) Nucleic Acids Res. 24: 3546-3551.

6. Fogg, et al. (2002). Nat Struct Biol. 9(12): 922-927.7. Borns, M. and Hogrefe, H. (2000) Strategies. 13: 12.

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