Tools for PCR

AMPLIFY—PURIFY—ANALYZE

2

PCR reagents from AffymetrixAffymetrix offers a wide selection of USB® PCR tools for robust, accurate, PCR applications to amplify and purify DNA.

AMPLIFY—We offer the reliable products you need for end point, real-time, hot start, long and accurate PCR, multiplex, qPCR, as well as direct PCR for difficult sample types. USB products for PCR provide the consistent performance you’d expect to achieve quality results.

PURIFY—The ExoSAP-IT® family of products are the single-step enzymatic solution for PCR purifica-tion without sample loss. Save time and money by eliminating spin columns.

ANALYZE—Our large selection of enzymes provides you with tools you need for the analysis of downstream applications. Many of the buffers and stock solutions used to analyze PCR products are available in a ready-to-use form.

At Affymetrix, we have everything you need for PCR. We test and re-test our reagents to ensure that they work reliably the first time, every time. We can also provide custom or bulk enzymes, formu-lated to your specific requirements.

Visit us at our website at www.usb.affymetrix.com for additional product information, technical ref-erences, and protocols.

Table of contents

AmplifyPCR product selection guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Taq polymerase product selection guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4End point PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4End point direct PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6End point hot start PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7End point RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Ultapure nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Real-time qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Real-time reverse transcription PCR (qRT-PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

PurifyPCR product cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14PCR purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Nucleic acid purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

AnalyzeEnzymes for Next Generation sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Sequenase enzyme and kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Thermostable sequencing enzymes and kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Modifying enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21PCR related reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

OEM, custom, and bulk reagent solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

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AMPLIFY

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USB® Taq DNA Polymerase [74165]

Taq PCR Master Mix (2X) [71162]

RubyTaq PCR Master Mix (2X) [71191]

FideliTaq™ DNA Polymerase [71180]

FideliTaq PCR Master Mix (2X) [71182]

VeriQuest® Taq DNA Polymerase [71170]

HotStart-IT® Taq DNA Polymerase [71195]

HotStart-IT Taq Master Mix (2X) [71196]

HotStart-IT FideliTaq DNA Polymerase [71155]

HotStart-IT FideliTaq PCR Master Mix (2X) [71156]

VeriScript™ Reverse Transcriptase [78070]

M-MLV Reverse Transcriptase [78306]

AMV Reverse Transcriptase [70041]

FideliTaq RT-PCR Master Mix (2X) [71185]

Tth DNA Polymerase [70052]

First-Strand cDNA Synthesis Kit for Real-Time PCR [75780]

VeriQuest SYBR® Green qPCR Master Mix (2X) [75600]

VeriQuest SYBR Green qPCR Master Mix with Fluorescein (2X) [75665]

VeriQuest Probe qPCR Master Mix (2X) [75650]

VeriQuest Probe qPCR Master Mix, No Reference Dye (2X) [75660]

VeriQuest Fast SYBR Green qPCR Master Mix (2X) [75690]

VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein (2X) [75675]

VeriQuest Fast Probe qPCR Master Mix (2X) [75680]

VeriQuest Fast Probe qPCR Master Mix, No Reference Dye (2X) [75685]

VeriQuest SYBR Green One-Step qRT-PCR Master Mix (2X) [75705]

VeriQuest SYBR Green One-Step qRT-PCR Master Mix with Fluorescein (2X) [75715]

VeriQuest Probe One-Step qRT-PCR Master Mix (2X) [75700]

VeriQuest Probe One-Step qRT-PCR Master Mix, No Reference Dye (2X) [75710]

MagniTaq® Multiplex PCR Master Mix [71199]

RubyTaq™ DNA Polymerase [71190]

VersaTaq™ Direct PCR Polymerase (Inhibition Resistant) [71002]

RT-PCR Master Mix (2X) [78370]

PCR product selection guide

Let our simple chart below help you find the optimal reagent for your research needs. Navigate our PCR product portfolio by application to view your options.

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AMPLIFY

Product name Product no. Description Properties Exonuclease activity Proof-reading activity

USB® Taq DNA Polymerase

74165 Thermostable Taq DNA polymerase for routine PCR up to 6 kb. Provided with 10X PCR reaction buffer.

1. Source: Recombinant2. Concentration: 5 units/µl3. Functionally tested in standard PCR

5'→3' No

VersaTaq Direct PCR Polymerase

71002 Mutant Taq DNA polymerase resistant to common PCR inhibitors, allowing for direct PCR amplification, thus eliminating the need for costly extraction and purification steps. Provided with 10X PCR reaction buffer.

1. Source: Recombinant (E. coli)2. Concentration: 1.0 ± 0.2 μg/μl, determined by OD2803. Resistance to potent PCR inhibitors in a wide range of end

point PCR applications: – Whole blood (up to 40%) – Feces – Blood spots on sample – Urine collection cards – Animal tissue – Buccal swabs – Plant tissue – Soil – Crude extracts

5’→3’ No

RubyTaq DNA Polymerase

71190 Taq DNA polymerase containing tracking dye for easy-to-see, easy-to-load convenience. Provided with 10X PCR reaction buffer and MgCl2 for optimization.

1. Source: Recombinant (E. coli)2. Concentration: 1 unit/µl3. Inert dyes: A magenta dye runs ~500 bp (on 2% gels) or ~1500 bp

(on 0.8% gels), and a yellow dye runs smaller than 10 bp.4. Functionally tested in standard PCR

5'→3' No

FideliTaq DNA Polymerase

71180 Combination of Taq DNA polymerase and a high-fidelity, proofreading polymerase for long and accurate PCR. Provided with 10X PCR reaction buffer and MgCl2 for optimization.

1. Source: Recombinant (E. coli)2. Concentration: 5 units/µl3. Fidelity 6X greater than standard Taq4. Generates 3:1 A-tailed to blunt ends5. Functionally tested to PCR amplify a 20.7 kb product

5’→3’ 3’→5’

Yes

HotStart-IT FideliTaq DNA Polymerase

71155 Hot start FideliTaq DNA polymerase containing a novel SSB protein that prevents primer dimers and non-specific amplification during reaction set-up for highly specific, long and accurate PCR. Provided with 10X PCR reaction buffer and MgCl2 for optimization.

1. Source: Recombinant (E. coli)2. Concentration: 2.5 units/µl3. No additional heat step required for activation like with other

hot start technologies4. Fidelity 6X greater than standard Taq5. Generates 3:1 A-tailed to blunt ends6. Functionally tested to prevent primer dimers and PCR amplify

a 20.7 kb product

5’→3’ 3’→5’

Yes

HotStart-IT Taq DNA Polymerase

71195 Hot start Taq DNA polymerase containing a novel SSB protein that prevents primer dimers and non-specific amplification during reaction set-up for highly specific PCR. Provided with 10X PCR reaction buffer and MgCl2 for optimization.

1. Source: Recombinant (E. coli)2. Concentration: 1.25 units/µl3. No additional heat step required for activation like with other

hot start technologies4. Functionally tested to prevent primer dimers in standard PCR

5'→3' No

MagniTaq Multiplex PCR Master Mix (2X)

71199 Multiplex master mix with a chemically-modified hot start DNA polymerase for highly sensitive and specific simultaneous amplification of even the most difficult templates (including FFPE) with minimal optimization.

1. Concentration: 2X2. Activation step: 5 minutes at 95°C3. Advanced buffer formulation that includes dNTPs and MgCl2

for minimal optimization requirements4. Validated performance for simultaneous amplification of at

least 20 targets up to 1 kb in length.

5'→3' No

VeriQuest Taq DNA Polymerase

71170 Chemically-modified hot start Taq DNA polymerase that prevents primer dimers and non-specific amplification during reaction set-up for highly sensitive and specific PCR. Provided with 10X PCR reaction buffer and MgCl2 for optimization.

1. Source: Recombinant (E. coli)2. Concentration: 5 units/µl3. Activation step: 10 minutes at 95°C4. Functionally tested in standard PCR

5’→3’ No

Taq polymerase product selection guide

Review our chart below to help you find the optimal Taq polymerase for your research needs.

End point PCR

USB Taq DNA Polymerase (5 units/μl)Thermostable Taq DNA polymerase for routine PCR up to 6 kb. Provided with 10X PCR reaction buffer.74165 50 units 400 units 2,000 units 4,000 units (2 x 2,000 units) 20,000 units

Taq PCR Master Mix, 2X (50 μl reaction volume)Supplied as a convenient 2X pre-mixed formulation containing Taq DNA Polymerase, Ultrapure nucleotides, and reaction buffer optimized for a wide variety of PCR applications.71162 100 reactions

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RubyTaq PCR Master Mix (2X)(50 μl reaction volume)Convenient 2X pre-mixed formulation containing Taq DNA Polymerase, Ultrapure nucleotides, reaction buffer, and two inert dyes.71191 100 reactions 500 reactions

FideliTaq™ DNA Polymerase (5 units/μl)Combines high quality recombinant Taq DNA Polymerase with a high-fidelity, proofreading polymerase. Includes 10X PCR buffer and 25 mM MgCl2 for optimization.71180 50 units 250 units 1,000 units 5 x 250 units 5,000 units

FideliTaq PCR Master Mix, 2X(50 μl reaction volume)Ready-to-use mix that combines Taq DNA Polymerase, a high-fidelity, proofreading polymerase, and Ultrapure nucleotides in a proprietary buffer formulation.71182 100 reactions

FideliTaq PCR Master Mix Plus (50 μl reaction volume)Includes FideliTaq PCR Master Mix (PN 71182) plus Ultrapure PCR-Qualified H2O and MgCl2 solution.71183 100 reactions

End point PCR (continued)

Taq Master Mix Plus(50 μl reaction volume)Contains Taq PCR Master Mix, 25 mM MgCl2 and PCR-Qualified H2O.71163 100 reactions

Tth DNA Polymerase(5 units/μl)Suitable for PCR in the presence of Mg2+ ions. It also has an intrinsic reverse transcriptase activity in the presence of Mn2+ ions. The reverse transcription activity using RNA as a template to prepare cDNA may be achieved at high temperatures, thereby eliminating problems related to sec-ondary structure.70052 200 units 1,000 units

RubyTaq™ DNA Polymerase(1 unit/μl)Contains high-quality Taq DNA Polymerase with two inert dyes that serve as tracking dyes in gel electropho-resis. Includes 10X PCR buffer and 25 mM MgCl2 for optimization.71190 50 units 250 units 1,000 units 5,000 units

RubyTaq DNA Polymerase visualization during gel electro-phoresis. RubyTaq Polymerase in PCR does not require the use of additional loading buffer. Simply load onto an agarose gel directly after cycling. During electrophoresis, RubyTaq separates into 2 col-ors, magenta (runs between 500 bp [2% gels] and 1500 bp [0.8% gels]) and yellow (runs less than 10 bp). The indicated volumes of RubyTaq DNA Polymerase were run on a 1% TAE agarose gel.

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End point direct PCR: Skip DNA extraction

VersaTaq™ Direct PCR Polymerase(25 μl reaction volume)In contrast to standard Taq DNA polymerase, VersaTaq Direct PCR Polymerase can amplify DNA directly from samples, which eliminates costly extraction and purification steps, saving time and enabling amplification from limited quantities of DNA.

– Whole blood (up to 40%) – Feces– Blood spots on sample – Urine

collection cards – Animal tissue– Buccal swabs – Plant tissue– Soil (humic acid) – Crude extracts

71002 50 reactions 200 reactions 500 reactions 2 x 500 reactions

Amplification of targets in presence of bile salts by VersaTaq Direct PCR Polymerase. Targets were amplified from 5 ng of genomic DNA in the presence of varying concentrations of bile salts. Lanes 1-2: NUMB 455 bp; Lanes: 3-4 NUMB 967 bp; Lanes: 5-6 HRES1 488 bp. 1 M betaine soil added to reactions with high GC content. Lanes 7-8: 110 bp lambda targets were amplified from 5 ng of lambda DNA in the presence of crude extract feces. Lane 8 amplification was also in the presence of RNAlater®.

Amplification of targets directly from mouse tail by VersaTaq Direct PCR Polymerase. Targets were amplified from 1 mm mouse tail clip added directly into PCR reaction. Lane 1: NUMB 483 bp; Lane 2: NUMB 1.8 kb.

M 1 2

Mouse Tail (length) M 1 mm

500 bp-

2000 bp-

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

500 bp -

2000 bp -

Blood (%)

M

10 40 10 40 10Anticoagulant E H C E H C E H C E H C E H C

E: Na-EDTAH: Na-HeparinC: Na-Citrate

Amplification of targets directly from human whole blood. Targets were amplified from different concentrations of human whole blood in the presence of varying types of anticoagulants. Lanes 1-6: NUMB 455 bp; Lanes 7-12: NUMB 1.8 kb; Lanes 13-15: HRES1 488 bp - corresponding band is an 81% GC amplicon. 1 M betaine added to reactions with high GC targets.

Amplicon GC %

M

48% 35% 81%25 75 25 75

500 bp-

2000 bp-

Bile salts (µg)

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End point hot start PCR

HotStart-IT® Taq DNA Polymerase(1.25 units/μl)Hot start Taq DNA polymerase containing a novel SSB protein that prevents primer dimers and non-specific amplification during reaction set-up for highly specific PCR. Includes 10X PCR buffer and 25 mM MgCl2 for optimization.71195 50 units 250 units 1,000 units 5 x 250 units 5,000 units

HotStart-IT Taq Master Mix, 2X(50 μl reaction volume)Convenient 2X pre-mixed formulation containing HotStart-IT Taq DNA Polymerase and Ultrapure nucleotides in a proprietary reaction buffer.71196 25 reactions 100 reactions 500 reactions

HotStart-IT Binding Protein (≥10 mg/ml)Active component in primer sequestration technique. Sufficient for 200 x 25 μl reactions.71194 400 μg

HotStart-IT FideliTaq DNA Polymerase (2.5 units/μl)Hot start FideliTaq DNA polymerase containing a novel SSB protein that prevents primer dimers and non-specific amplification during reaction set-up for highly specific, long and accurate PCR. Includes 10X PCR reaction buffer and 15 mM MgCl2 for optimization.71155 50 units 250 units 1,000 units 5,000 units

HotStart-IT FideliTaq PCR Master Mix, 2XConvenient formulation that contains HotStart-IT FideliTaq DNA Polymerase and Ultrapure nucleotides in a proprietary reaction buffer.71156 25 reactions 100 reactions 500 reactions

VeriQuest® Taq DNA Polymerase(5 units/μl)Chemically-modified hot start Taq DNA polymerase that pre-vents primer dimers and non-specific amplification during reaction set-up for highly sensitive and specific PCR. Includes 10X PCR reaction buffer and MgCl2 for optimization.71170 50 units 250 units 1,000 units 5,000 units

PCR Reaction Preparation without Hot Start

• primers with self-complementary region

5 3

5 3

5 3

553

5 3

• primers hybridize to each other at low temperatures

PCR Reaction Preparation with USB Hot Start Method

5 3

5 3

53

5 3

5 3

53

• polymerases are recruited to hybrid• primer-dimers are produced• PCR Failure

• binding proteins interact with primers• primers are sequestered at low temperatures

• prevents non-specific hybridization and extension

• denaturation step inactivates binding proteins• primers bind to specific targets

• PCR Success

3Polymerase

Polymerase

• primers with self-complementary region

Binding Proteins

HotStart-IT method: primer sequestration

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End point RT-PCR

Enzymes:

VeriScript™ Reverse Transcriptase(200 units/µl)This reverse transcriptase is an engineered version of M-MLV with reduced RNase H activity and increased ther-mostability, allowing for first-strand cDNA synthesis with more full length cDNAs, up to 12.5 kb. VeriScript Reverse Transcriptase is active up to 50°C, which offers greater sensitivity and consistent performance for a wide range of RT applications.n Active over a wide temperature range. Recommended

incubation temperature of 42°C, but incubation tem-peratures up to 50°C may be used for RNA templates with difficult regions containing secondary structure or GC-rich regions.

n Robust cDNA synthesis is achieved with templates as long as 12.5 kb

n Compatible with oligo dT, random, or gene-specific priming

n Picogram level sensitivity in RT end point PCRn Efficient amplification in RT real-time PCR from as few

as 5 copies of synthetic RNA78070 4,000 units 10,000 units 4 x 10,000 units

amplicon length

T (°C)

0.5 kb 2.8 kb

42 45 48 50 55 42 45 48 50 55 42 45 48 50 55

6.6 kb

M M

100 bp -

500 bp -

1.0 kb -

2.0 kb -

4.0 kb -

12.0 kb -

HeLa total RNA (1 µg) was used in a 20 µl first-strand cDNA synthesis with 200 units of VeriScript Reverse Transcriptase. Reactions were incubated at the temperatures indicated on the figure for 30 minutes, followed by heat inactivation for 15 minutes at 70°C. RNA was removed by adding 1 µl (5 units) RNase H (PN 70054) and incubation at 37°C for 10 minutes, followed by heat inactivation for 10 minutes at 65°C. 10 ng cDNA was used in a 50 µl PCR reaction using FideliTaq DNA Polymerase (PN 71180) for 35 cycles. VeriScript Reverse Transcriptase is active over a wide temperature range. Incubation temperatures up to 50°C may be used for RNA templates with difficult regions containing secondary structures or GC-rich regions.

M-MLV Reverse Transcriptase(200 units/μl)This enzyme has a low RNase H activity that results in high yields of full length cDNA.78306 25,000 units 100,000 units

AMV Reverse Transcriptase (25 units/μl)Catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybrids; Synthesizes cDNA for PCR, clon-ing and hybridization probes.70041Y 200 units70041Z 1,000 units

One-step master mixes:

RT-PCR Master Mix, 2X(50 μl reaction volume)Includes M-MLV Reverse Transcriptase, Taq DNA Polym-erase, recombinant RNase Inhibitor, nucleotides, and mag-nesium in a novel RT-PCR buffer.78370 100 reactions

FideliTaq RT-PCR Master Mix, 2X(50 μl reaction volume)Includes M-MLV Reverse Transcriptase, FideliTaq DNA Polymerase, recombinant RNase Inhibitor, nucleotides, and magnesium in a novel RT-PCR buffer.71185 100 reactions

Kits:

One-Step RT-PCR Kit The One-Step RT-PCR Kit is designed for simpleRT-PCR in a one-tube format.n Streamlines and optimizes the RT and PCR stepsn Provides a starting point for analysis of new RNA targets78350 50 reactions

Two-Step RT-PCR Kit The Two-Step RT-PCR Kit is designed for flexibleRT-PCR in one- or two-tube formats.n Optimization of PCR independent of RTn Analysis of the expression of multiple genes in individual

RNA samples with oligo dT primers78355 50 RT reactions and 100 PCR reactions

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Multiplex PCR

MagniTaq® Multiplex PCR Master Mix, 2XSingle-tube, 2X multiplex PCR master mix with a chemi-cally-modified hot start DNA polymerase for highly sensi-tive and specific simultaneous amplification of at least 20 targets up to 1 kb in length of even the most difficult tem-plates (including FFPE) with minimal optimization.

1 10 100 1000 ng template DNA

1000900800700600

500

400

300

200

100

1252 1252

142 142

1500

1000

750

500

300

150

50

22-plex amplification of low amounts of gDNA. 22-plex amplification of gDNA targets with MagniTaq Multiplex PCR Master Mix. 1, 10, 100, and 1000 ng human genomic DNA input amounts (1 ng is about 100 cells) are added to the 22-plex notch-pathway single-copy gene primer set. Primer concentration is 0.2 µM and samples were loaded with SYBR® Green dye and run on an agarose gel.

MagniTaq Multiplex PCR Master Mix, 2X contains hot start MagniTaq DNA Polymerase, dNTPs, and MgCl2 in a propri-etary buffer formulation designed to enable multiplex PCR without lengthy optimization procedures.

71199 25 reactions 100 reactions

µM primers: 0.1 0.2 0.4

1000900800700600

500

400

300

200

100

1252

142

1500

1000

750

500

300

150

50

22-plex of a wide range of primer concentrations. MagniTaq Multiplex PCR Master Mix is effective over a wide range of primer concentrations. 100 ng human genomic DNA input amount is added to the 22-plex notch-pathway, single-copy gene primer set. Duplicate reactions were run using 0.1, 0.2 or 0.4 µM primer con-centrations. Samples were loaded with SYBR Green dye and run on an agarose gel.

Ultrapure nucleotides

dNTPs, Set of Four (dA, dC, dG, dT), 100 mM Solutions 4 dNTPs per pack77100 4 x 25 μmol (250 μl)

dNTPs, Set of Four (dA, dC, dG, dT), 100 mM Solutions4 dNTPs per pack77328 4 x 100 μmol

dNTPs, Set of Four (dA, dC, dG, dT), 100 mM Solutions4 dNTPs per pack77128 4 x 500 μmol

PCR Nucleotide Mix, 10 mM Solution77212 500 μl 2 x 500 μl

PCR Nucleotide Mix, 25 mM Solution77119 500 μl

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Real-time qPCR

Instrument compatibility with USB VeriQuest qPCR Master MixesUse this chart to determine which of our USB VeriQuest qPCR master mixes we recommend for your machine. Simply find your thermo cycler across the top row and then locate which of our master mixes are optimized for that instrument.

VeriQuest qPCR solutions:

Proven performance from a trusted source.Using a highly specific and sensitive master mix with a wide dynamic range is key for precise quantification and validation. VeriQuest qPCR reagents provide premium qPCR performance at affordable price points, allowing you to publish with confidence.n Single-tube, 2X master mixes for SYBR green or

TaqMan® probe assaysn 4°C storage up to 4 months—no need for freeze/thawn Performance on virtually any qPCR machine, fast cycling

formulations availablen Superior consistency, sensitivity, and assay linearity

VeriQuest Taq DNA Polymerase(5 units/μl)Chemically-modified hot start Taq DNA polymerase that prevents primer dimers and non-specific amplification during reaction set-up for highly sensitive and specific PCR. Includes 10X PCR reaction buffer and MgCl2 for optimization.71170 50 units 250 units 1,000 units 5,000 units

SYBR Green Mix Probe Mix Fast SYBR Green Mix Fast Probe Mix One-Step qRT-PCR SYBR Green Mix One-Step qRT-PCR Probe Mix

VeriQuest® SYBR® Green qPCR Master

Mix (2X) [75600]

VeriQuest SYBR Green qPCR Master

Mix with Fluorescein

(2X) [75665]

VeriQuest Probe qPCR Master Mix

(2X) [75650]

VeriQuest Probe qPCR

Master Mix, No Reference Dye (2X) [75660]

VeriQuest Fast SYBR Green qPCR Master

Mix (2X) [75690]

VeriQuest Fast SYBR Green qPCR Master

Mix with Fluorescein

(2X) [75675]

VeriQuest Fast Probe qPCR Master Mix

(2X) [75680]

VeriQuest Fast Probe qPCR

Master Mix, No Reference Dye (2X) [75685]

VeriQuest SYBR Green One-

Step qRT-PCR Master Mix

(2X) [75705]

VeriQuest SYBR Green One-Step qRT-PCR Master

Mix with Fluorescein

(2X) [75715]

VeriQuest Probe One-Step

qRT-PCR Master Mix

(2X) [75700]

VeriQuest Probe One-Step

qRT-PCR Master Mix, No Reference Dye (2X) [75710]

ABI Prism® 7000

ABI Prism 7700

ABI Prism 7900 HT*

ABI 5700

ABI 7300

ABI 7500*

ABI StepOne™/StepOne-Plus™*

ABI ViiA™ 7

Bio-Rad iCycler iQ®/iQ5

Bio-Rad iCycler MyiQ™

Bio-Rad CFX96™/CFX384™

Bio-Rad Opticon2™

Bio-Rad Chromo4™

Cepheid Smart Cycler®

Corbett Rotor-Gene™

Eppendorf Mastercycler® ep realplex

Fluidigm BioMark™

Illumina® Eco™*/Helixis Pixo™

Qiagen Rotor-Gene™ Q*

Roche Light Cycler® 1.0, 1.5, 2.0*

Roche LightCycler 480/1536*

Stratagene Mx3000P™*

Stratagene Mx3005P™*

Stratagene Mx4000™

TaKaRa TP-800™

* Instruments can be set to fast mode cycling when using fast mode master mixes. Indicates preferred kit for this cycler Indicates may also be used for this cycler

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AMPLIFY

VeriQuest Probe qPCR Master Mix, 2X with ROX(50 μl reaction volume)75650 40 reactions (1 ml) 200 reactions (5 ml) 400 reactions (2 x 5 ml) 1,000 reactions (5 x 5 ml) 2,000 reactions (10 x 5 ml)

Linear detection range of VeriQuest Probe qPCR Master Mix. Real-time amplification plot from a 10-fold dilution series of a GAPDH synthetic target with starting amounts of 1010 copies amplified in four replicate reactions using the ABI 7500 PCR System and GAPDH primer-probe set (FAM-BHQ®-1).

VeriQuest Fast Probe qPCR Master Mix, 2X with ROX(20 μl reaction volume)75680 100 reactions (1 ml) 500 reactions (5 ml) 1,000 reactions (2 x 5 ml) 2,500 reactions (5 x 5 ml) 5,000 reactions (10 x 5 ml)

First-Strand cDNA Synthesis Kit for Real-Time PCR Optimized for reverse transcription of RNA and produces first-strand cDNA template suitable for real-time PCR75780 50 reactions (20 μl)

(Continued on next page)

VeriQuest SYBR Green qPCR Master Mix, 2X with ROX(50 μl reaction volume)75600 40 reactions (1 ml) 200 reactions (5 ml) 400 reactions (2 x 5 ml) 1,000 reactions (5 x 5 ml) 2,000 reactions (10 x 5 ml)

Linear detection range of VeriQuest SYBR Green qPCR Master Mix. Real-time amplification plot from a 10-fold dilution series of a GAPDH synthetic target with starting amounts of 109 copies amplified in four replicate reactions using the ABI StepOne™ Real-Time PCR System.

References:

1. VanGuilder, H. D., Vrana, K. E., and Freeman, W. M. (2008) BioTechniques 44 (5), 619-626.

2. Longo, M. C., Berninger, M. S., and Hartley, J. L. (1990) Gene 93, 125-128.

3. Tewhey, R. et al. (2009) Nature Biotechnology 27, 1025-1031.

VeriQuest Fast SYBR Green qPCR Master Mix, 2X with ROX(20 μl reaction volume)75690 100 reactions (1 ml) 500 reactions (5 ml) 1,000 reactions (2 x 5 ml) 2,500 reactions (5 x 5 ml) 5,000 reactions (10 x 5 ml)

Real-time qPCR (continued)

For use with Applied Biosystems, Agilent, and Fluidigm instruments:

12

AMPLIFY

VeriQuest SYBR Green qPCR Master Mix with Fluorescein, 2X(50 μl reaction volume)75665 40 reactions (1 ml) 200 reactions (5 ml) 400 reactions (2 x 5 ml) 1,000 reactions (5 x 5 ml) 2,000 reactions (10 x 5 ml)

VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein, 2X(20 μl reaction volume)75675 100 reactions (1 ml) 500 reactions (5 ml) 1,000 reactions (2 x 5 ml) 2,500 reactions (5 x 5 ml) 5,000 reactions (10 x 5 ml)

GAPDH Kras

10 43 2 1 ng RNA

10 4 3 2 1 ng RNA

GAPDH2y = -3.3773x + 22.224

EPCR = 97.7%R² = 0.9955

Krasy = -3.2461x + 30.591

EPCR = 103.3%R² = 0.9993

151719212325272931

-0.2 0 0.2 0.4 0.6 0.8 1 1.2

Ct

log input

GAPDH2 Kras

High sensitivity and precision in limited target quantifica-tion. Amplification plot (left) and standard curve (right) from real-time PCR for a 1.33 to 10-fold dilution series of 10 ng to 1 ng of cDNAs reverse-transcribed from HeLa total RNA.

VeriQuest Probe qPCR Master Mix, No Reference Dye, 2X(50 μl reaction volume)75660 40 reactions (1 ml) 200 reactions (5 ml) 400 reactions (2 x 5 ml) 1,000 reactions (5 x 5 ml) 2,000 reactions (10 x 5 ml)

VeriQuest Fast Probe qPCR Master Mix, No Reference Dye, 2X(20 μl reaction volume)75685 100 reactions (1 ml) 500 reactions (5 ml) 1,000 reactions (2 x 5 ml) 2,500 reactions (5 x 5 ml) 5,000 reactions (10 x 5 ml)

Real-time qPCR (continued)

For use with BioRad, Eppendorf, and Roche instruments:

13

AMPLIFY

Real-time reverse transcription PCR (qRT-PCR)

For use with BioRad, Eppendorf, and Roche instruments:

VeriQuest SYBR Green One-Step qRT-PCR Master Mix with Fluorescein, 2X(50 μl reaction volume)The 2X master mix contains 100X RT Enzyme Mix and 2X qRT-PCR Mix for RNA quantification with SYBR Green. The 100X RT Enzyme Mix is a blend of reverse transcriptase and RNase Inhibitor. The 2X qRT-PCR Mix contains chem-ically-modified VeriQuest Taq DNA Polymerase, ultrapure nucleotides, SYBR Green I, and Fluorescein Passive Refer-ence Dye in an optimized buffer formulation for quantita-tive, real-time PCR detection with SYBR Green.75715 40 reactions (1 ml) 200 reactions (5 ml)

VeriQuest Probe One-Step qRT-PCR Master Mix, No Reference Dye, 2X(50 μl reaction volume)The 2X master mix contains reverse transcriptase, RNase inhibitor, our chemically modified VeriQuest Taq DNA Polymerase, and ultrapure nucleotides all in an optimized buffer composition.75710 40 reactions (1 ml) 200 reactions (5 ml)

For use with Applied Biosystems, Agilent, and Fluidigm instruments:

VeriQuest SYBR Green One-Step qRT-PCR Master Mix, 2Xwith ROX(50 μl reaction volume)The 2X master mix contains 100X RT Enzyme Mix and 2X qRT-PCR Mix for RNA quantification with SYBR Green. The 100X RT Enzyme Mix is a blend of reverse transcriptase and RNase Inhibitor. The 2X qRT-PCR Mix contains hot start VeriQuest Taq DNA Polymerase, ultrapure nucleo-tides, SYBR Green I, and ROX Passive Reference Dye in an optimized buffer formulation for quantitative, real-time PCR detection with SYBR Green.75705 40 reactions (1 ml) 200 reactions (5 ml)

VeriQuest Probe One-Step qRT-PCR Master Mix, 2Xwith ROX(50 μl reaction volume)The 2X master mix contains reverse transcriptase, RNase inhibitor, our chemically modified VeriQuest Taq DNA polymerase, ROX Passive Reference Dye, and ultrapure nucleotides all in an optimized buffer composition.75700 40 reactions (1 ml) 200 reactions (5 ml)

13.0

19.8 23.4

26.8

30.4 33.2

14.0

20.9

24.4 27.8

30.7 32.9

13.3

20.0

23.6 26.9

30.5 33.5

0

5

10

15

20

25

30

35

40

100 ng 10 ng 1 ng 100 pg 10 pg 1 pg 100 fg

Ct

HeLa Total RNA

ABI TaqMan Fast Virus 1-Step

ABI TaqMan RNA-to-CT™ 1-Step

VeriQuest One-Step Probe

16.5 17.5 16.6

slope EPCR R2

ABI TaqMan Fast Virus 1-Step -3.4087 96.50% 0.9994

ABI TaqMan RNA-to-CT 1-Step -3.2142 104.70% 0.9953

VeriQuest One-Step Probe -3.4057 96.62% 0.9998

Product comparison—VeriQuest Probe One-Step qRT-PCR Master Mix. Ct values, R2, and PCR efficiency from real-time PCR for a 10-fold dilution series of 100 ng to 100 fg HeLa total RNA amplified in duplicate reactions with VeriQuest Probe One-Step qRT-PCR Master Mix, ABI TaqMan Fast Virus 1-Step Master Mix, and ABI TaqMan RNA-to-CT 1-Step Kit using the ABI 7500 Fast Real-Time PCR System and GAPDH primers and probe (Fam-BHQ) in standard mode with 15 minute RT reaction at 50°C.

Exceptional results with performance comparisons.

14

PURIFY

PCR product cleanup

Which ExoSAP-IT PCR cleanup reagent is right for you?

ExoSAP-IT products offer a unique, one-tube, one-step, enzymatic process for PCR product cleanup.All ExoSAP-IT reagents provide 100% sample recovery with no loss of PCR products regardless of the fragment size. This PCR cleanup method removes excess primers and dNTPs and does not interfere with downstream applications. Achieve superior results with ExoSAP-IT reagent—improve accuracy with higher yields and full PCR product recovery.

Use our comparison chart below to determine which formulation is best for your next experiment.

ExoSAP-IT® ExpressExoSAP-IT reagentOur original formulation

HT ExoSAP-IT Fast High-Throughput For automated liquid handlers

Protocol time 5 minutes 30 minutes 14 minutes

Format Single tube 8-tube strips 96-well plate

Single tube Single tube 8-tube strips96-well plate

Throughput level Low to high; Recommended for processing any sample size

Low to mid; Recommended for processing 1-96 samples at a time

High; Recommended for processing ≥ 96 samples at a time

Platform Single- or multi-channel pipette, automated liquid handling platforms

Single-channel pipette Automated liquid handling platforms

Freezes at -20°C No No Yes

Stability -20°C for up to 2 years -20°C for up to 2 years -20°C for up to 2 years;Once thawed, stable at 4°C for 1 month and RT for 2 days

How ExoSAP-IT reagent works. Treat 5 μl of PCR product with 2 μl of ExoSAP-IT reagent. Treatment is carried out at 37°C followed by an incubation period at 80°C to completely inactivate both enzymes. Once these contaminants are removed, your PCR products are ready for down-stream applications such as sequencing (Sanger/NGS), fragment analysis, SNP analysis, in vitro transcription, or single base extension.

37°C, 4 min for treatment 80°C, 1 min to inactivate

Add ExoSAP-IT®

Express Reagent*

* Representative of all ExoSAP-IT formulations. Incubation times differ for ExoSAP-IT Express, original ExoSAP-IT, and HT ExoSAP-IT Fast reagents.

OverviewExoSAP-IT reagents are a patented mixture of Exonuclease I combined with Shrimp Alkaline Phosphatase (SAP) in a spe-cially formulated buffer that removes excess primers and dNTPs following a PCR reaction. Exonuclease I removes residual single-stranded primers and any extraneous single-stranded DNA produced during PCR. SAP removes the remaining dNTPs from the PCR mixture which may interfere with subsequent reactions.

15

PURIFY

ExoSAP-IT Express reagent: PCR cleanup in 5 minutesOur one-step ExoSAP-IT Express reagent ensures quality sequencing results in a fraction of the time.n 5 minute protocol—Fastest turnaround time to resultsn One-tube, one-step PCR cleanup—Add directly to

PCR productn Novel enzyme technology—Enzymes are irreversibly

inactivated in just 1 minute at 80°Cn Conserve PCR samples—100% recovery and no loss of

PCR products, regardless of amplicon lengthn Scaleable—Treat PCR reaction volumes from 5 µl to 5 Ln Eliminate spin columns or magnetic beads—Signifi-

cantly decrease time and expense while increasing yield

Conserve PCR samples—simple one-step, 100% recoveryThe ExoSAP-IT Express enzymatic cleanup method minimizes errors by reducing your protocol to a single pipette step, allowing for automated or manual processing. ExoSAP-IT Express reagent outperforms the competition by ensuring 100% recovery of all amplicon sizes, both short and long.

Table 1. DNA recovery after purificationPCR size Agencourt AMPure® XP beads ExoSAP-IT reagent

86 bp 10% 100%

103 bp 12% 100%

545 bp 63% 100%

1007 bp 88% 100%

PCR product cleanup (continued)

High quality—accurate resultsCompared to alternative PCR cleanup methods, ExoSAP-IT Express reagent provides accurate and consistent results in just 5 minutes. This unique, highly stable, one-tube solution allows for 100% recovery of DNA and longer read lengths for greater confidence, consistency, and accuracy. Treated PCR products produced superior results when treated with ExoSAP-IT Express reagent.

Fastest PCR cleanup methodOur researchers have engineered a heat labile Exonuclease I, enabling rapid enzyme inactivation in only 1 minute. This innovation from the original ExoSAP-IT reagent lets scientists continue their workflow without disruptions. ExoSAP-IT Express reagent offers faster turnaround times and improved efficiency of resources while delivering superior sequencing results.

75001 40 μl 20 reactions75001 200 μl 100 reactions75001 1 ml 500 reactions75001 4 × 1 ml 2,000 reactions75001 10 ml 5,000 reactions75001 1 ea 480 reactions × 8-tube strip

Sequencing results when treated with ExoSAP-IT Express reagent. Sequencing of a 1 kb PCR product treated with ExoSAP-IT Express. ExoSAP-IT Express treatment prior to sequencing eliminates miscalls and improves sequencing scores (numbers and bars above sequence; >60, probability of error ≤0.0001%). Sequence shown is approximately 400 bases from primer binding site.

Compare cleanup methods. Use of ExoSAP-IT Express reagent eliminates spin columns, magnetic beads, sedimentations, filtrations, and gel purifications. With a 5 minute protocol, ExoSAP-IT Express reagent is the fastest and easiest method for PCR cleanup, minimizing pipetting errors or contamination.

(Continued on next page)

16

PURIFY

PCR product cleanup (continued)

ExoSAP-IT PCR Product CleanupConsidered the gold standard in enzymatic PCR cleanup, ExoSAP-IT PCR Product Cleanup has been cited in over 10,000 publications. Ideal cleanup for Sanger sequencing, TA cloning, in vitro transcription, and SNP analysis.n 30 minute cleanup protocoln Recommended for processing 1-96 samples at a timen Best cost per reaction value

Rapid PCR product cleanup protocolExoSAP-IT reagent requires only one pipette step and two incubations. Just add ExoSAP-IT reagent to the PCR prod-uct and within 30 minutes sequencing or SNP analysis can be performed.

Simple: single-stepThe method is designed to require a minimum of ‘hands-on’ time. Enzymatic removal of excess primers and unin-corporated nucleotides occurs in one easy step by using ExoSAP-IT reagent in a single tube or microtiter well. Only simple pipette transfers are required, therefore, many samples can be processed at once, either manually or with robotics.

No sample lossUse of ExoSAP-IT reagent eliminates all gel or column puri-fications, sedimentations, filtrations, beads, and/or mag-netic separations(1). There is 100% recovery of both short and long PCR products with ExoSAP-IT reagent.

HES-1 numb NRAGE numb M prepostprepostprepostprepost

12kb–

2kb–

1kb–

100bp–

ExoSAP-IT treatment of PCR products with no sample loss. Single-copy targets were amplified from human genomic DNA. HES-1 (125 bp), numb (455 bp), NRAGE (1.55 kb), and numb (4.6 kb) were loaded on a 1.5% agarose gel before (pre) and after (post) ExoSAP-IT treatment. M is the DNA marker lane. Note that a vari-ety of PCR product sizes can be treated with ExoSAP-IT, even a 125 bp fragment, with no sample loss.

USBExoSAP-ITreagent

Spincolumn

2 miscalls

Fluorescent sequencing results of a 100 bp pUC18 PCR fragment sequenced with a -20 Fwd primer using fluores-cent sequencing reagents. PCR cleanup performed with: (a) ExoSAP-IT; (b) a column designed for PCR cleanup. Base miscalls in (b) are due to inherently low yields of short PCR products when using columns.

30 40

GCATcGCCTGCTAAGCT-GCC

GCATCGCCTGCTAAGCT-GCC

GCAN-GCCNGCTAAGCTCGCC

4 miscalls

USB ExoSAP-ITreagent

Alternative enzymatic reagent

Fluorescent sequencing results of a 1007 bp PCR product. A 1007 bp fragment was amplified and treated with ExoSAP-IT reagent (above) or an alternate enzymatic reagent (below) and sequenced. Pherograms revealed no miscalls with ExoSAP-IT reagent but four miscalls at position 25, 26, 30, and 39 with the alternate enzymatic reagent. An amount of 5 µl of PCR product was treated with 2 µl ExoSAP-IT reagent or the alternate enzymatic method per its protocol. Note: Two of the four miscalls are frame-shifts.

78250 40 μl 20 reactions78200 200 μl 100 reactions78201 1 ml 500 reactions78202 4 × 1 ml 2,000 reactions78205 10 ml 5,000 reactions

References:1. Dugan, K. A., Lawrence, H. S., Hares, D. R., Fisher, C. L. and Budowle B.

(2002) J. Forensic Sci 47, 811-818.

2. Hanke, M. and Wink, M. (1994) BioTechniques 17, 858-860.

3. Mu, J., Duan, J., Makova, K., Joy, D., Huynh, C., Branch, O., Li, W. and Su, X. (2002) Nature 418, 323-326.

4. Silva, Jr., W. A., Costa, M. C. R., Valente, V., De Freitas Sousa, J., Paco-Larson, M. L., Espreafico, E. M., Camargo, S. S., Monteiro, E., De Jesus, A., Holanda, M. A., Zago, M. A., Simpson, A. J. G. and Neto, E. D. (2001) BioTechniques 30, 537-542.

5. Werle, E., Scneider C., Renner, M., Volker, M. and Fiehn, W. (1994) Nucl. Acids Res. 22, 4354-4355.

17

PURIFY

HT ExoSAP-IT Fast High-Throughput PCR Product CleanupHT ExoSAP-IT Fast High-Throughput PCR Product Cleanup is an alternative formulation of the original ExoSAP-IT reagent specifically designed for the unique requirements of high-throughput, automated platforms and multi-channel pipettes.

Like the original formulation, HT ExoSAP-IT Fast reagent is a mixture of Exonuclease I and Shrimp Alkaline Phosphatase for the enzymatic removal of excess primers and dNTPs following a PCR reaction. When PCR amplifi-cation is complete, any unconsumed dNTPs and primers remaining in the PCR product mixture will interfere with downstream applications. The ExoSAP-IT products remove these contaminants. HT ExoSAP-IT Fast reagent is scalable for PCR cleanup from a single tube up to a 384-well plate PCR reaction.

HT ExoSAP-IT Fast reagent is over 50% faster than the original HT ExoSAP-IT reagent with a 14 minute total pro-tocol time. Add it directly to the PCR product and incubate at 37°C for 7 minutes. After PCR treatment, inactivate it by simply heating to 80°C for another 7 minutes.

This product is ideal for high volume labs that require quick sample turn around. With such a brief and simple protocol, proper sample cleanup never needs to be sacrificed due to processing or instrument time constraints. Ensure the high-est quality Sanger sequencing results while keeping sample processing moving quickly and efficiently.

PCR products cleaned up with HT ExoSAP-IT Fast reagent prior to automated sequencing displayed superior sequenc-ing results compared to the untreated equivalent.

PCR product cleanup (continued)

Why HT ExoSAP-IT Fast reagent?n 14 minute cleanup protocoln 100% recovery and only 5 μl of PCR product neededn One simple pipetting stepn Formulated and formatted for automated liquid han-

dling platforms and multi-channel pipettesn Stable at 4°C for one month and at room temperature

for 2 days

HT ExoSAP-IT Fast reagent displayed excellent stability in all formats tested including vials, 8-tube strips, and 96-well plates. The product showed no loss in function after 10 freeze thaw cycles, and is stable at variable temperatures for extended periods of time compared to enzyme mix equivalents. HT ExoSAP-IT Fast reagent offers a decreased viscosity and is ideal for high-throughput assays where automated liquid handling platforms are utilized and large volumes of samples are processed in successive testing runs.

78595 20 rxn 20 reactions78595 1 ea 480 reactions × 8-tube strip78595 1 pk 5,760 reactions (12 × 8- tube strips in a tray)78595 4 pk 23,040 reactions (48 × 8- tube strips in a tray)78595 1000 rxn 1,000 reactions (2 ml)78595 5000 rxn 5,000 reactions (10 ml)78595 1 pl 1,920 reactions (96-well plate, 40 µl per well)78595 6 pl 11,520 reactions (6 × 96- well plate, 40 µl per well)

18

PURIFY

PCR purification

PCR Product Pre-Sequencing Kit The PCR Product Pre-Sequencing Kit uses a novel enzy-matic cleanup method to pre-treat PCR products prior to sequencing without any subsequent purification or sepa-ration steps. The two hydrolytic enzymes used in this kit, Shrimp Alkaline Phosphatase (SAP) and Exonuclease I, effectively remove excess dNTPs and primers present in the final PCR product reaction mixture. The enzymes are conveniently added to an aliquot of the PCR product mix-ture, incubated at 37°C, and then inactivated at 80°C. The result is a cleaner PCR product, free from excessive nucleo-tides and primers, ready to be sequenced with standard sequencing reagents.70995 100 reactions70996 500 reactions70997 2,000 reactions

PrepEase® PCR Purification 96-Well Plate Kits (Ultrafiltration) Designed for high-throughput purification of PCR products using 96-well plates.78761 10 x 96-well plates78762 50 x 96-well plates

Nucleic acid purification

DNA isolation

PrepEase DNA Cleanup Kits Binding capacity up to 15 μgDesigned for the purification of DNA from reactions or for buffer exchange78758 50 preps78759 250 preps

PrepEase Gel Extraction Kits Binding capacity up to 15 μgDesigned for purifying DNA fragments from agarose gels. Also suited for PCR product purification.78756 50 preps78757 250 preps

RNA purification

PrepEase RNA Spin KitsBinding capacity up to 100 μgDesigned for the isolation of total RNA from cellsand tissue78766 50 preps78767 250 preps

Plasmid purification

PrepEase Plasmid Gravity-Flow Column KitsDesigned to obtain low to high copy plasmid DNA from bacterial cultures.

PrepEase Midi Plasmid KitBinding capacity up to 100 μgCulture volume up to 30 ml78706 100 preps

PrepEase Maxi Plasmid KitBinding capacity up to 500 μgCulture volume up to 150 ml78711 50 preps

PrepEase Quick MiniSpin Plasmid KitDesigned for very rapid purification78742 250 preps

PrepEase MiniSpin Plasmid KitDesigned for high yield purification78737 250 preps

19

Enzymes for Next Generation sequencing

Next Generation Sequencing (NGS) is becoming an essen-tial component of molecular biology research to allow scientists to quickly and economically sequence DNA and RNA. We provide a range of USB enzymes for your NGS workflow. All of these reagents can be produced in large

ANALYZE

quantities or formulated to custom specifications to meet your requirements.n Stringent testing and QC analysisn Consistent lot-to-lot performancen Quality products from our enzymology experts

Key USB enzymes: Key features

Relevance in

amplicon-based NGS

Relevance in adapter ligation-

based NGS

End repair and

A-tailing

Conversion of RNA to

DNA

Phosphory-lation of

DNA

Adapter ligation

MagniTaq Multiplex PCR Master Mix

Simultaneous amplification of ≥20 targets from difficult templates

ü

Exonuclease-Free Klenow Klenow fragment lacking 3’-5’ exonuclease activity ü ü

rDNase I, RNase-Free Free of RNases, sufficient removal of DNA contaminants ü ü ü

T4 Gene 32 Protein Enhances cDNA synthesis and T4 DNA Polymerase ü ü ü

SSB, E. coli Stimulates DNA sequencing polymerase and enables longer read lengths

ü ü

Exonuclease I Removal of ssDNA primers in between PCR amplifications, irreversible heat inactivation

ü

Exonuclease VII Only bi-directional exonuclease with single-stranded speci-ficity, no requirement for divalent cations

ü

T4 Polynucleotide Kinase Phophorylation of nucleic acid 5’-ends and hydroxylation of 3’-ends. Use with OptiKinase™ for least bias and optimal sequencing coverage

ü ü

OptiKinase Reduces 5’ end bias in kinase reactions and drives 3’ phosphatase activity of T4 PNK for enhanced sequencing coverage

ü ü

T4 DNA Ligase, High Concentration

Efficient adapter ligation to DNA fragments ü ü ü

NGS workflow solutions

ExoSAP-IT Express PCR Product Cleanup

Gold standard PCR cleanup with 100% recovery for efficient NGS library preparation

ü ü

PrepEase Genomic DNA Isolation Kit

Rapid isolation of gDNA for NGSü ü

Change-IT™ Multiple Mutation Site Directed Mutagenesis Kit

Efficient mutagenesis for gene function reverse genetics studies by NGS ü ü

20

Mutagenesis

Change-IT™ Multiple Mutation Site Directed Mutagenesis KitThe Change-IT Multiple Mutation Site Directed Mutagenesis Kit is designed to create single or multiple oligonucleotide-directed sequence changes in plasmids. It also allows for efficient mutagenesis for gene function reverse genetics studies by NGS.78480 20 reactions

Sequenase enzyme and kitsSequenase Version 2.0 DNA Polymerase Sequenase Version 2.0 is highly processive, incorporates nucleotide analogs (dlTP, thio-dNTPs, dideoxy-NTPs, etc.), is not impeded by secondary structures, and can carry out strand displacement synthesis. It is an excellent enzyme for dideoxy-sequencing and is useful in other applications, especially where the absence of associated exonuclease activity is desirable.70775Y 200 units70775Z 1,000 units

Sequenase Version 2.0 DNA Sequencing Kit This kit features Sequenase Version 2.0 DNA Polymerase which is highly processive and able to effectively incorpo-rate nucleotide analogs for sequencing. Use of the specially formulated buffers and mixes included in the kit will maxi-mize yield of sequence information.70770 100 reactions

Sequenase Quick Denature Plasmid DNA Sequencing Kit This kit offers two simple and efficient denaturation meth-ods that do not require precipitation of plasmid DNA. Both methods, Glycol/Glycerol and Alkali/Acid, allow for rapid generation of denatured template DNA suitable for use with standard Sequenase Version 2.0 DNA Polymerase. In addition, this kit contains the 7-deaza-dGTP nucleotide mixes which provide stronger band intensities for resolving sequencing gel compressions.70140 100 reactions

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Thermostable sequencing enzymes and kitsUSB CycleSeq™ Thermostable DNA Polymerase(32 units/µl)USB CycleSeq Thermostable DNA Polymerase is a ther-mostable, DNA polymerase that is exonuclease-free and incorporates ddNTPs with a marked level of efficiency over standard thermostable DNA polymerases. This allows for more even and easy-to-read sequence band patterns, making sequence anomalies easier to identify. It is useful for cycle sequencing (Sanger sequencing), as it produces sequence data with uniform band intensities, allowing for longer and more accurate sequence reads. It is also useful for primer extension protocols during SNP genotyping.79200 1,000 units 10,000 units

USB CycleSeq Polymerase sequencing results are comparable to GE Thermo Sequenase. Cycle sequencing using a fluorescently-labeled primer and 300 ng pUC19 template DNA was performed using either Thermo Sequenase DNA Polymerase or CycleSeq Thermostable DNA Polymerase. Analysis of both sequences shows equivalent quality, read length, and band intensity.

Thermo Sequenase Cycle Sequencing Kit This kit allows for equally efficient incorporation of both ddNTPs and dNTPs in cycle sequencing reactions resulting in very uniform band intensities.78500 100 reactions

Thermo Sequenase Dye Primer Manual Cycle Sequencing Kit Kit is designed to be used with fluorescent dye-labeled primers and high-resolution fluorescence scanners.79260 50 reactions

21

ANALYZE

Modifying enzymes

AMV Reverse Transcriptase (15 units/μl)Catalyzes the polymerization of DNA using template DNA, RNA, or RNA:DNA hybrids; Synthesizes cDNA for PCR, cloning and hybridization probes.70041Y 200 units70041Z 1,000 units

Calf Intestinal Alkaline Phosphatase (CIAP) (20 units/μl)Utilized for dephosphorylation of 5’-phosphorylated ends of DNA or RNA for subsequent labeling.70034Y 1,500 units

Exonuclease IEffective for removal of ssDNA primers in between PCR amplifica-tions; irreversible heat inactivation.Standard concentration 10 units/μl70073Z 2,500 units70073X 5,000 unitsHigh concentration 20 units/μl 72073 5,000 units

Exonuclease VII (10 units/μl)Only bi-directional exonuclease with single-stranded specificity, no requirement for divalent cations.70082Y 200 units70082Z 1,000 units

Exonuclease-Free Klenow (10 units/μl)Klenow fragment lacking 3’-5’ exonuclease activity.70057Y 125 units70057Z 750 units70057 2,500 units

M-MLV Reverse Transcriptase (200 units/μl)This enzyme has a low RNase H activity that results in high yields of full length cDNA.78306 25,000 units 100,000 units

OptiKinase™ (10 units/μl)Reduces 5’ end bias in kinase reactions and drives 3’ phosphatase activity of T4 PNK for enhanced sequencing coverage.78334X 500 units78334Y 1,000 units78334Z 2,500 units

rDNase I, RNase-Free (10 units/μl)Free of RNases; sufficient removal of DNA contaminants78411 1,000 units 2,500 units

Shrimp Alkaline Phosphatase (SAP) (1 unit/μl)Used to treat unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.78390 500 units 1,000 units 5,000 units

Single-stranded DNA Binding Protein (SSB) (5 μg/μl)SSB stimulates DNA sequencing polymerase and enables longer read lengths.70032Y 100 μg70032Z 500 μg

T4 DNA LigaseEfficient adapter ligation to DNA fragmentsStandard concentration, 1 unit/μl70005Y 100 units70005X 500 units70005 2,000 unitsHigh concentration, 10 units/μl70042X 500 units70042 2,000 units

T4 Gene 32 ProteinT4 Gene 32 Protein enhances cDNA synthesis and T4 DNA Polymerase.Standard concentration, 5 μg/μl70029Y 100 μg70029Z 500 μgHigh concentration, ≥ 10 μg/μl74029Y 300 μg74029Z 1,000 μg

Tth DNA Polymerase (5 units/μl)Suitable for PCR in the presence of Mg2+ ions. It also has an intrin-sic reverse transcriptase activity in the presence of Mn2+ ions. The reverse transcription activity using RNA as a template to prepare cDNA may be achieved at high temperatures, thereby eliminating problems related to secondary structure.70052 200 units 1,000 units

T4 Polynucleotide Kinase (30 units/μl)Phophorylation of nucleic acid 5’-ends and hydroxylation of 3’-ends. Use with OptiKinase for least bias and optimal sequenc-ing coverage70031Y 500 units70031Z 1,000 units70031X 2,500 units

VeriScript Reverse Transcriptase(200 units/µl)This reverse transcriptase is an engineered version of M-MLV with reduced RNase H activity and increased thermostability, allowing for first-strand cDNA synthesis with more full length cDNAs, up to 12.5 kb. VeriScript Reverse Transcriptase is active up to 50°C which offers greater sensitivity and consistent performance for a wide range of RT applications.78070 4,000 units 10,000 units 4 x 10,000 units

22

PCR related reagents

ANALYZE

Agarose - Hi-Res™, Separation ≤1000 bpUltrapure10132 25 gm 100 gm 500 gm

Agarose - LE Ultrapure32802 25 gm 100 gm 250 gm 500 gm 1 kg

Agarose - Low Melt, Separation, ≤1000 bpGenetic Performance Certified™Ultrapure32829 25 gm 100 gm

Agarose - Low Melt, Separation, ≥1000 bpGenetic Performance CertifiedUltrapure32830 25 gm 100 gm

Agarose - Separation ≥500 bp Genetic Performance CertifiedUltrapure75817 25 gm 100 gm 250 gm 500 gm

Betaine, 5 M Solution Ultrapure77507 1.5 ml 5 x 1.5 ml 10 ml

DNA Ladder, 1 kb Plus76714 500 µl

DNA Ladder, 100 bp76712 500 µl

Fluorescein Passive Reference DyeUltrapure75767 500 µl

Ligate-IT™ Rapid Ligation Kit78400 25 reactions78410 100 reactions

Magnesium Chloride (MgCl2), 1 M Solution Ultrapure78641 10 x 1 ml 100 ml

Mineral Oil Ultrapure71600 10 ml 25 ml 1 L

PCR Markers, 50—2000 bp76710 250 μl

RapidRun™ Agarose Buffer, 20X SolutionUltrapure77523 1 L 5 L

ROX Passive Reference Dye Ultrapure75768 500 μl

Sodium Acetate (NaOAc) 3M Solution, pH 5.5Ultrapure75897 100 ml 500 ml

TAE Buffer, 10X SolutionUltrapure75904 1 L 5 L

TBE Buffer, 5X Solution Ultrapure75891 1 L 5 L

TE Buffer, 1X Solution Ultrapure75893 10 x 1 ml 100 ml 500 ml

TE Buffer, 50X Solution Ultrapure75834 100 ml 500 ml

Tris/HCl, 1M Solution, pH 7.0 Ultrapure22637 100 ml 500 ml 1 L

Tris/HCl, 1M Solution, pH 7.5Ultrapure22639 100 ml 500 ml 1 L

Tris/HCl, 1M Solution, pH 8.0 Ultrapure22638 100 ml 500 ml 1 L

Water, Nuclease-FreeUltrapure71786 10 x 1 ml 100 ml 500 ml 1 L 5 L

23

OEM, custom, and bulk reagent solutions We provide custom and OEM solutions to meet your growing needs.USB has been helping diagnostic, biotech, and pharma-ceutical companies succeed in the life sciences industry for over 40 years. We can confidently assist you with custom reagent manufacturing, dispensing, labeling, and kitting of your critical reagent components. Optimize your use of key staff and limited resources by outsourcing production of your finished goods or key components.

Custom and OEM solutions: Enzymes, biochemicals and detergentsn Custom or bulk enzymes, formulated to your special

requirementsn Special fill sizes/concentrations/formulations of over

2,500 catalog productsn OEM labeling of individual components, or complete kit

packaging and labeling with your brandn General Purpose Reagents (GPR) and IVD reagents and

assays to your exacting standardsn Customer-specific quality control assaysn Our experts will work with you at your site and/or from

our reagents center of excellence

ISO 13485 compliant processesn Rigorous quality system suited for your diagnostic

requirementsn Comprehensive documentationn Rigorous change control capabilitiesn We welcome audits of our manufacturing facilities

Get started today!Let us help you with your next project. Contact us at 888-362-2447 (216-765-5000 outside the US), or email us at USBCustom@affymetrix.com to get started.

Please visit our website at usb.affymetrix.com for interna-tional distributor contact information.

Product portfolioHere’s a sampling of our more than 2,500 customizable products:

EnzymesLigases, kinases, phosphatases, polymerases, and Sequenase

Convenience reagentsPre-mixed buffers and stock solutions

PCR reagentsExoSAP-IT PCR Cleanup reagent in multiple formats and formulations, SSB binding protein, DNA polymerases, and

exo-free klenow

NucleotidesdNTPs, PCR nucleotide mixes, ddNTPs, and NTPs

BiochemicalsAgaroses, acrylamides, amino acids, sugars, substrates, and vitamins

Affymetrix, Inc.USB® Products26111 Miles RoadCleveland, Ohio 44128Tel: 800-321-9322 | 216-765-5000Fax: 800-535-0898 | 216-464-5075USBcustomerserv@affymetrix.comusb.affymetrix.com

Affymetrix UK Ltd.USB® ProductsVoyager, Mercury Park,Wycombe Lane, Wooburn Green,High Wycombe HP10 0HH, UKTel: +44 (0)1628 55 2600Fax: +44 (0)1628 55 2675USBcustomerserveurope@affymetrix.com

Affymetrix Pte Ltd.USB® Products7 Gul Circle, #2M-01Keppel Logistics BuildingSingapore 629563Tel: +65 6395 7310Fax: +65 6395 7300USBcustomerserv@affymetrix.com

© 2015, 2016 Affymetrix, Inc. All rights reserved. Rev 07/15/16P/N USB04621 Rev. 7

For Research Use Only. Not for use in diagnostic procedures.

Please visit our website at usb.affymetrix.com for international distributor contact information.

Affymetrix, USB, ExoSAP-IT, HotStart-IT, MagniTaq, PrepEase, and VeriQuest are registered trademarks of Affymetrix, Inc. Change-IT, CycleSeq, FideliTaq, Genetic Performance Certified, Hi-Res, Ligate-IT, OptiKinase, RapidRun, RubyTaq, VeriScript, and VersaTaq are trademarks of Affymetrix, Inc. Please see affymetrix.com/trademarks for a complete list of Affymetrix trademarks. All other trademarks are the property of their respective owners.

Illumina is a registered trademark of Illumina, Inc. Affymetrix, Inc. is not affiliated with, associated with, sponsored by, or endorsed by Illumina, Inc.

ExoSAP-IT PCR Product Cleanup product line—These products are covered by patents owned by Affymetrix, including US Patent Nos. 6,379,940 and 6,387,634 and corresponding patents in Canada and Japan.

MagniTaq Multiplex PCR Master Mix—Patent pending.

Taq DNA Polymerase—Purchase of this product conveys to the purchaser the limited, non-transferable right under certain patents to use only the purchased amount of product for the purchaser’s own internal research. No right under any other patent claims or for any other use is conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting Out-licensing at Thermo Fisher Scientific, 5791 Van Allen Way, Carlsbad, California 92008; outlicensing@lifetech.com.

SYBR Green Products—Use of these products are covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. The purchase of these products includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. This product is also provided under an agreement between Molecular Probes, Inc. and Affymetrix and the manufacture, use, sale or import of this product is subject to one or more US Patents and corresponding international equivalents, owned by Molecular Probes, a Life Technologies Corporation company. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product for real-time PCR conducted by the buyer, for use in research and development, which excludes use in performing testing, analysis or screening services. The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party, whether or not such product or its components are resold for use in research and development, and shall not use this product or its components or materials made using this product or its components for therapeutic or prophylactic purposes. For information on purchasing a license to this product for purposes other than set forth above, contact Molecular Probes, Inc., Business Development, 29851 Willow Creek Road, Eugene, OR 97402, USA. Tel: (541) 465-8300, Fax: (541) 335-0354.