Roche PCR Reagents and PCR Protocols

• Roche PCR Reagents Selection Guide for Routine and Hot Start PCR
• Roche PCR Reagents Selection Guide for High Fidelity, Difficult Templates, and Long PCR Amplification
  
• Taq DNA Polymerase and Standard PCR Protocol
• FastStart™ Taq DNA Polymerase and Master Mix
• FastStart™ High Fidelity PCR System
• Expand™ High Fidelity PCR System and Expand™ High Fidelity^PLUS PCR System
• Pwo SuperYield DNA Polymerase
• GC-RICH PCR System for Difficult Template PCR
• Expand™ Long Range, dNTPack
• Products for PCR Optimization

Roche PCR Reagents Selection Guide for Routine and Hot Start PCR

PCR Application	Routine PCR	Hot Start	Multiplex/Sequencing
Your Enzyme of Choice:	Taq DNA Polymerase	FastStart™ Taq DNA Polymerase	FastStart™ High Fidelity PCR System
Experience	Consistency	Specificity	Accuracy
Amplicon Size	up to 3 kb	up to 3 kb	up to 5 kb
Specificity
Sensitivity
Robustness
Accuracy vs. Taq	1x	1x	4x
Carryover Prevention	yes	yes	yes
Units/50 μL	1.25	2	2.5
Molecular Cloning	TA cloning	TA cloning	TA cloning
Enzyme	Taq DNA Polymerase, 5 U/μL Taq DNA Polymerase, 1 U/μL	FastStart™ Taq DNA Polymerase, 5 U/μL	FastStart™ High Fidelity PCR System
Enzyme, GMP Grade	Taq DNA Polymerase, GMP Grade, 5 U/μL
dNTPack	Taq DNA Polymerase, dNTPack, 5 U/μL Taq DNA Polymerase, dNTPack, 1 U/μL	FastStart™ Taq DNA Polymerase, dNTPack, 5 U/μL	FastStart™ High Fidelity PCR System, dNTPack
Master Mix	PCR Master	FastStart™ PCR Master

Roche PCR Reagents Selection Guide for High Fidelity, Difficult Templates, and Long PCR Amplification

* Requiring carryover prevention and 6x higher fidelity? Choose Expand™ High FidelityPLUS PCR System. ** For amplicon sizes of 20-35 kb choose Expand™ 20 kbPLUS PCR System.

PCR Application	Wide Variety/Easy Access	High Fidelity	Difficult Templates	Long Range
Your Enzyme of Choice:	Expand™ High Fidelity PCR System	Pwo SuperYield DNA Polymerase	GC-RICH PCR System	Expand™ Long Range
Experience	Robustness	Fidelity	Reliability	Versatility
Amplicon Size	up to 5 kb	up to 3 kb	up to 5 kb	5 to 25 kb**
Specificity
Sensitivity
Robustness
Accuracy vs. Taq	3x	18x	3x	3x
Carryover Prevention	no*	no	no	no
Units/50 μl	2.6	2.5	2	3.5
Molecular Cloning	TA cloning	Blunt end cloning	TA cloning preferred over blunt end cloning	TA cloning
Enzyme	Expand™ High Fidelity PCR System Expand™ High FidelityPLUS PCR System	Pwo SuperYield DNA Polymerase	GC-RICH PCR System
dNTPack	Expand™ High Fidelity PCR System, dNTPack Expand™ High FidelityPLUS PCR System, dNTPack	Pwo SuperYield DNA Polymerase, dNTPack	GC-RICH PCR System, dNTPack	Expand™ Long Range, dNTPack
Master Mix	High Fidelity PCR Master	Pwo Master

Cutting-edge science shouldn’t have to compromise for the sake of economy. Since the introduction of PCR technology, Roche has provided the gold standard in PCR reagents. Our advanced isolation, purification, and manufacturing techniques ensure that you receive not only the clearest, most reliable results, but also convenience and affordability to fit any laboratory budget.

• Reliability – Achieve the results you deserve, from lot to lot, tube to tube, and experiment to experiment.
• Performance – Combine premium enzymes plus PCR-Grade Nucleotides in convenient dNTPacks for enhanced sensitivity and yield.
• Consistency – Maximize experimental performance using master mixes that only require the addition of primers and template.

PCR-Grade Nucleotides ensure optimal performance.

PCR success is highly dependent on your selection of both enzymes and nucleotides. Roche PCR enzymes are available in convenient dNTPacks, which include premixed solutions of additive-free sodium salt nucleotides. These function tested PCR-Grade Nucleotides are manufactured by enzymatic synthesis and purified by a unique process and are an ideal contributor to the quality of your PCR results.

• Purity – Choose nucleotides free of modified bases, tetraphosphates, or pyrophosphate contaminants according to current quality procedures typical to chemically synthesized nucleotide preparations to improve your PCR’s sensitivity.
• Stability – Lengthen shelf life and improve reaction stability using dNTPs supplied at an optimal pH to maximize yield.

Taq DNA Polymerase and Standard PCR Protocol

Affordable PCR characterized by robust amplification with minimal template requirements.

Roche Taq DNA Polymerase is produced under GMP conditions. This highly purified enzyme passes several stringent tests for functionality and purity, ensuring reliable, consistent results with every lot. For additional information, please view our standard PCR protocol.

• Obtain reliable, reproducible results with high lot-to-lot consistency
• Eliminate testing of each new lot – we do it for you
• Combine dUTP incorporation with Uracil-DNA Glycosylase to prevent PCR cross-contamination

Figure 1. Lot-to-lot consistency ensures reproducible results. Five different lots of Taq DNA Polymerase were tested for the ability to amplify a 0.5 kb fragment of lambda DNA. Reliable, consistent results are obtained with every lot of Roche Taq DNA Polymerase that was tested.

The Taq DNA Polymerase, GMP Grade belongs to the family of high-performance, validated amplification enzymes and is manufactured using evaluated production, quality control, and filling procedures.

Amplicon Size	Specificity	Sensitivity	Robustness	Accuracy vs. Taq	Carryover Prevention	Units /50 µL	Molecular Cloning
Up to 3 kb				1x	yes	1.25	TA cloning

Product	SKU	Pack Size
Taq DNA Polymerase | Enzyme + Buffer
5 U/μL	11 146 173 001	500 U for up to 1,000 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
	11 418 432 001	1,000 U (4 × 250 U) for up to 2,000 reactions of 20 μL final volume, each containing 0.5 Taq DNA Polymerase
	11 596 594 001	2,500 U (10 × 250 U) for up to 5,000 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
	11 435 094 001	5,000 U (20 × 250 U) for up to 10,000 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
1 U/μL	11 647 687 001	1,000 U (4 × 250 U) for up to 2,000 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
Taq DNA Polymerase, dNTPack | Enzyme + Buffer + dNTP Set
5 U/μL	04 728 866 001	100 U for up to 200 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
	04 728 874 001	500 U (2 × 250 U) for up to 1,000 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
	04 728 882 001	1,000 U (4 × 250 U) for up to 2,000 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
	04 728 904 001	2,500 U (10 × 250 U) for up to 5,000 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
	04 728 858 001	5,000 U (20 × 250 U) for up to 10,000 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
1 U/μL	04 738 225 001	250 U for up to 500 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
	04 738 241 001	1,000 U (4 × 250 U) for up to 2,000 reactions of 20 μL final volume, each containing 0.5 U Taq DNA Polymerase
PCR Master | Ready-to-use Formulation; Enzyme, Buffer, dNTPs
	11 636 103 001	1 kit for up to 200 PCR reactions of 50 μL final reaction volume, each containing 2.5 U Taq DNA Polymerase

FastStart™ Taq DNA Polymerase and Master Mix

Choose the polymerase researchers trust, as with qPCR and the LightCycler^® Systems.

FastStart™ Taq DNA Polymerase is a thermostable, chemically modified form of recombinant Taq DNA polymerase. Due to its modification, FastStart™ Taq DNA polymerase is only activated at high temperatures, making it the ideal enzyme when your assay’s amplification is delayed.

• Obtain higher specificity, sensitivity, and yield. Hot start PCR improves PCR performance, making PCR setup even easier.
• Use a convenient robotic setup. The complete reaction mix is stable for 24 hours at room temperature.
• Prevent PCR carryover. Combine dUTP incorporation with Uracil-DNA Glycosylase to prevent PCR cross-contamination.

Figure 4. Amplification of the human erythropoietin gene from 3-300 ng cDNA after immediate (E0) and 24 hours after reaction setup at room temperature (E24).

Amplicon Size	Specificity	Sensitivity	Robustness	Accuracy vs. Taq	Carryover Prevention	Units /50 µL	Molecular Cloning
Up to 3 kb				1x	yes	2 µL	TA cloning

Product	SKU	Pack Size
FastStart™ Taq DNA Polymerase | Enzyme + Buffer
5 U/μL	12 032 929 001	500 U (2 × 250 U) for up to 250 reactions of 50 μL final volume, each containing 2 U FastStart Taq DNA Polymerase
	12 032 937 001	1,000 U (4 × 250 U) for up to 500 reactions of 50 μL final volume, each containing 2 U FastStart Taq DNA Polymerase
	12 032 945 001	2,500 U (10 × 250 U) for up to 1,250 reactions of 50 μL final volume, each containing 2 U FastStart Taq DNA Polymerase
	12 032 953 001	5,000 U (20 × 250 U) for up to 2,500 reactions of 50 μL final volume, each containing 2 U FastStart Taq DNA Polymerase
FastStart™ Taq DNA Polymerase, dNTPack | Enzyme + Buffer + dNTP Set
5 U/μL	04 738 314 001	100 U for up to 50 reactions of 50 μL final volume, each containing 2 U FastStart Taq DNA Polymerase
	04 738 357 001	500 U (2 × 250 U) for up to 250 reactions of 50 μL final volume, each containing 2 U FastStart Taq DNA Polymerase
	04 738 381 001	1,000 U (4 × 250 U) for up to 500 reactions of 50 μL final volume, each containing 2 U FastStart Taq DNA Polymerase
	04 738 403 001	2,500 U (10 × 250 U) for up to 1,250 reactions of 50 μL final volume, each containing 2 U FastStart Taq DNA Polymerase
	04 738 420 001	5,000 U (20 × 250 U) for up to 2,500 reactions of 50 μL final volume, each containing 2 U FastStart Taq DNA Polymerase
FastStart™ PCR Master | Ready-to-use Formulation; Enzyme, Buffer, dNTPs
	04 710 436 001	2 × 1.25 mL for up to 250 reactions of 20 μL final reaction volume
	04 710 444 001	8 × 1.25 mL for up to 1,000 reactions of 20 μL final reaction volume
	04 710 452 001	10 × 5 mL for up to 5,000 reactions of 20 μL final reaction volume

FastStart™ High Fidelity PCR System

Multiplexing and sequencing stringent hot start and improved fidelity is key in complex endpoint PCR applications, such as multiplexing or sequencing. Choose FastStart™ High Fidelity PCR System for complex hot start PCR up to 5 kb with higher fidelity, as required for multiplexing and sequencing. It is up to six times more accurate compared to both standard Taq DNA polymerase and FastStart™ Taq DNA Polymerase.

• Achieve excellent multiplex performance with fast, high-quality results
• Experience six-fold higher fidelity compared to Taq and FastStart™ Taq DNA Polymerase
• Use the supplied PCR additive (DMSO) when amplifying difficult templates

Figure 5. Sensitivity test in 18-plex (74 bp – 470 bp) PCR. Set of 18 multiplexed primers was applied to various concentrations of human genomic DNA.

Amplicon Size	Specificity	Sensitivity	Robustness	Accuracy vs. Taq	Carryover Prevention	Units /50 µL	Molecular Cloning
Up to 5 kb				6x	yes	2.5	TA cloning

Product	SKU	Pack Size
FastStart™ High Fidelity PCR System | Enzyme + Buffer
	03 553 400 001	500 U (2 × 250 U) for up to 200 reactions of 50 μL final volume, each containing 2.5 U FastStart Taq DNA Polymerase
	03 553 361 001	2,500 U (10 × 250 U) for up to 1,000 reactions of 50 μL final volume, each containing 2.5 U FastStart Taq DNA Polymerase
FastStart™ High Fidelity PCR System, dNTPack | Ready-to-use Formulation; Enzyme, Buffer, dNTPs
	04 738 284 001	125 U for up to 50 reactions of 50 μL final volume, each containing 2.5 U FastStart Taq DNA Polymerase
	04 738 292 001	500 U (2 × 250 U) for up to 200 reactions of 50 μL final volume, each containing 2.5 U FastStart

Expand™ High Fidelity PCR System and Expand™ High FidelityPLUS PCR System

Expand™ High Fidelity PCR reagents are designed for robust amplification across a broad range of assays and amplicon types, consisting of an enzyme blend of Taq DNA Polymerase and a polymerase with proofreading activity for robust PCR. Choose the Expand™ High Fidelity^PLUS PCR System for robust PCR up to 5 kb with PCR carryover prevention.

• Use one enzyme for all applications. The Expand™ High Fidelity PCR System robustly amplifies a wide variety of templates ensuring high yield, fidelity, and flexibility.
• Detect amplification products that were previously undetectable and avoid false negatives.
• Achieve successful PCR results from small quantities of template DNA.

The Expand™ High Fidelity^PLUS PCR System is ideal for robust high-fidelity applications, such as cloning and labeling of DNA fragments with radioactively or nonradioactively modified nucleotides. In addition, combine dUTP incorporation with uracil-DNA glycosylase to prevent PCR cross-contamination.

Figure 6. Comparison of Expand™ High FidelityPlus PCR System with four commercially available polymerase mixes. Various amounts (ng) of human genomic DNA were used to amplify a 4.8 kb fragment from the tissue plasminogen activator (tPA) gene, in accordance with each manufacturer's recommended conditions. Expand High FidelityPLUS PCR System produces the best specificity, sensitivity, and yield, even from as little as 1 ng human genomic DNA.

Supplier A: Mixture of Taq DNA polymerase (deleted at N-terminus), a proofreading polymerase, and a hot start antibody.
Supplier B and D: Mixture of Taq DNA polymerase and a proofreading polymerase.
Supplier C: Mixture of Taq DNA polymerase, a proofreading polymerase, and an enhancing factor.

Amplicon Size	Specificity	Sensitivity	Robustness	Accuracy vs. Taq	Carryover Prevention	Units /50 µL	Molecular Cloning
Up to 5 kb				3x	no*	2.6	TA cloning

Product	SKU	Pack Size
Expand™ High Fidelity PCR System | Enzyme + Buffer
	11 732 641 001	100 U for up to 40 reactions of 50 μL final volume, each containing 2.6 U enzyme blend
	11 732 650 001	500 U (2 × 250 U) for up to 200 reactions of 50 μL final volume, each containing 2.6 U enzyme blend
	11 759 078 001	2,500 U (10 × 250 U) for up to 1,000 reactions of 50 μL final volume, each containing 2.6 U enzyme blend
Expand™ High Fidelity PCR, dNTPack | Enzyme + Buffer + dNTP Set
	04 738 250 001	100 U for up to 40 reactions of 50 μL final volume, each containing 2.6 U enzyme blend
	04 738 268 001	500 U (2 × 250 U) for up to 200 reactions of 50 μL final volume, each containing 2.6 U enzyme blend
	04 738 276 001	2,500 U (10 × 250 U) for up to 1,000 reactions of 50 μL final volume, each containing 2.6 U enzyme blend
High Fidelity PCR Master | Ready-to-use Formulation; Enzyme, Buffer, dNTPs
	12 140 314 001	1 kit for up to 500 reactions of 20 μL final reaction volume

Product	SKU	Pack Size
Expand™ High FidelityPLUS PCR System | Enzyme + Buffer
	03 300 226 001	500 U (2 × 250 U) for up to 500 reactions of 20 μL final volume, each containing 1 U enzyme blend
	03 300 234 001	2,500 U (10 × 250 U) for up to 2,500 reactions of 20 μL final volume, each containing 1 U enzyme blend
Expand™ High FidelityPLUS PCR System, dNTPack | Enzyme + Buffer + dNTP Set
	04 743 725 001	125 U for up to 125 reactions of 20 μL final volume, each containing 1 U enzyme blend
	04 743 733 001	500 U (2 × 250 U) for up to 500 reactions of 20 μL final volume, each containing 1 U enzyme blend

Pwo SuperYield DNA Polymerase

High fidelity PCR is required in applications where sequence accuracy is crucial without sacrificing yield. Use Pwo SuperYield DNA Polymerase to obtain high yields of PCR product with consistent high fidelity. Choose maximum fidelity and avoid sequence errors, base exchanges, and frame shifts when combined with a proofreading reverse transcriptase, such as in the Transcriptor High Fidelity cDNA Synthesis Kit. The combination is ideal for applications such as cloning, site-directed mutagenesis, and gene expression analysis.

• Achieve excellent fidelity and high yields. 18-fold higher fidelity compared to Taq DNA Polymerase, without optimization.
• Obtain high performance with difficult templates. The GC-RICH Solution enables amplification of GC-rich DNA.
• Reduce working steps in cloning. Perform enzyme digests directly in Pwo SuperYield PCR mix.

Figure 7. Improved accuracy in RT-PCR. Combination of the proofreading reverse transcriptase, Transcriptor High Fidelity Reverse Transcriptase (Roche), and a proofreading polymerase, Pwo SuperYield DNA Polymerase (Roche) for amplification. Data compared to a commonly used M-MuLV reverse transcriptase and Taq DNA polymerase. Error rate was determined after reverse transcription and 25 PCR cycles using the 454 Sequencing System. The error rates for the Roche enzymes was the mean value of four independent experiments in which at least 3.1 × 106 bases were sequenced. For M-MuLV reverse transcriptase and Taq DNA Polymerase, 4.5 × 106 bases were sequenced. The accuracy is represented as error rate -1.

Amplicon Size	Specificity	Sensitivity	Robustness	Accuracy vs. Taq	Carryover Prevention	Units /50 µL	Molecular Cloning
Up to 3 kb				18x	no	2.5	blunt end cloning

Product	SKU	Pack Size
Pwo Super Yield DNA Polymerase | Enzyme + Buffer
	04 340 850 001	500 U (2 × 250 U) for up to 200 reactions of 50 μL final volume, each containing 2.5 U Pwo SuperYield DNA Polymerase
Pwo Super Yield DNA Polymerase, dNTPack | Enzyme + Buffer + dNTP Set
	04 743 750 001	100 U for up to 40 reactions of 50 μL final volume, each containing 2.5 U Pwo SuperYield DNA Polymerase
Pwo Master | Ready-to-use Formulation; Enzyme, Buffer, dNTPs
	03 789 403 001	2.5 ml (10 × 250 μL) for up to 100 reactions of 50 μL final reaction volume, each containing 2.5 U Pwo SuperYield DNA Polymerase

GC-RICH PCR System for Difficult Template PCR

Choose the GC-RICH PCR System, a blend of a proofreading polymerase and Taq DNA Polymerase to power through templates that are difficult or impossible to amplify with other polymerases or other blends of polymerases and additives. The enhanced processivity of the blend and the unique GC-RICH Resolution Solution are combined to deliver superior performance.

• Amplify difficult templates, including GC-rich targets and repetitive sequences
• Use the supplied PCR Grade Water and optimized reagents, including the GC-RICH Resolution Solution
• Amplify DNA fragments up to 5 kb

Figure 8. Successfully amplify GC-rich templates with the GC-RICH PCR System. Amplification of a 264 bp template (74% GC content) within the human ApoE gene using the GC-RICH PCR System or the Expand High Fidelity PCR System.

Result: The GC-RICH PCR System amplifies the GC-rich fragment with high specificity and yield using GC-RICH Resolution Solution.

Amplicon Size	Specificity	Sensitivity	Robustness	Accuracy vs. Taq	Carryover Prevention	Units /50 µL	Molecular Cloning
Up to 5 kb				3x	no	2	TA cloning preferred over blunt end cloning

Product	SKU	Pack Size
GC-RICH PCR System | Enzyme + Buff
	12 140 306 001	100 U for up to 50 reactions of 50 μL final volume, each containing 2 U enzyme blend
GC-RICH PCR System, dNTPack | Enzyme + Buffer + dNTP Set
	04 743 784 001	100 U for up to 50 reactions of 50 μL final volume, each containing 2 U enzyme blend

Expand™ Long Range, dNTPack

Rely on this next-generation system from Roche, the company that pioneered long-template PCR. Choose Expand™ Long Range dNTPack for consistent amplification of PCR products of 5 to 25 kb from genomic DNA. When amplifying fragments longer than 20 kb, use Expand™ 20 kb^PLUS PCR System. This unique system features an optimized buffer and enzyme blend mixture.

• Consistently amplify long templates up to 25 kb with high specificity, yield, and increased fidelity.
• Save time and resources with a convenient and flexible kit. Use one buffer for all fragment sizes and use the supplied DMSO and MgCl₂ solution to fine-tune your reaction.
• Use for all long-template applications, obtaining high yields of only the desired fragment.
• Order the cost-effective dNTPack which also includes premixed PCR-Grade nucleotides.

Figure 9. Amplification of various genomic templates using Expand™ Long Range, dNTPack

Amplicon Size	Specificity	Sensitivity	Robustness	Accuracy vs. Taq	Carryover Prevention	Units /50 µL	Molecular Cloning
5 to 25 kb				3x	no	3.5	TA cloning

Product	SKU	Pack Size
Expand™ Long Range, dNTPack | Enzyme + Buffer + dNTP Set
	04 829 034 001	175 U for up to 125 reactions of 20 μL final volume, each containing 1.4 U enzyme blend
	04 829 042 001	700 U for up to 500 reactions of 20 μL final volume, each containing 1.4 U enzyme blend
	04 829 069 001	3,500 U (5 × 700 U) for up to 2,500 reactions of 20 μL final volume, each containing 1.4 U enzyme blend

* For amplicon sizes of 20-35 kb: choose Expand 20 kbPLUS PCR System.

Product	SKU	Pack Size
Expand™ 20 kbPLUS PCR System | Enzyme + Buffer
	11 811 002 001	200 U for up to 40 reactions of 50 μL final volume, each containing 5 U enzyme blend

Products for PCR Optimization

Product	SKU	Pack Size
PCR Optimization Kit	11 636 138 001	1 kit for up to 100 one-step or up to 50 two-step optimization assays of 100 μL final reaction volume
Uracil-DNA Glycosylase	11 444 646 001	100 U
Uracil-DNA Glycosylase, heat-labile	11 775 367 001	100 U
	11 775 375 001	500 U
T4 Gene 32 Protein	10 972 983 001	100 µg
	10 972 991 001	500 µg
PCR Buffer Set	11 699 121 001	2 x 2 mL (2 × 1 mL of each solution)
PCR Buffer without MgCl2 10x conc	11 699 105 001	3 × 1 mL
MgCl2 Stock Solution	11 699 113 001	3 × 1 mL
Water, PCR Grade	03 315 843 001	100 mL (4 vials of 25 mL)
	03 315 932 001	25 mL (25 vials of 1 mL)
	03 315 959 001	25 mL (1 vial of 25 mL)
Strip PCR Tubes and Caps	11 667 009 001	125 strips (8 tubes/strip)