novagen PCR ENZYMES O VERVIEW

2 www.novagen.com PCR ENZYME S ELECTION G UIDE Enzyme PCR Product Elongation Specificity Fidelity GC-rich Yield PCR Product Size Rate Templates Ends

KOD HiFi DNA Polymerase < 6 kbp 120 bases/s ● ■■blunt KOD Hot Start DNA Polymerase < 21 kbp 120 bases/s ■■ ■■blunt KOD XL DNA Polymerase < 30 kbp 120 bases/s ● ■■■Mixed (blunt and 3'-dA) NovaTaq™ DNA Polymerase < 5 kbp 60 bases/s ●● ●3'-dA NovaTaq Hot Start DNA Polymerase < 5 kbp 60 bases/s ■ ● ▲ 3'-dA NovaTaq DNA Polymerase + Taq Antibody < 5 kbp 60 bases/s ■ ● ▲ 3'-dA

● Satisfactory ▲ Good ■ Excellent

Novagen offers a complete selection of enzymes and kits for PCR, featuring KOD HiFi DNA Polymerase. This unique proofreading enzyme, isolated from the extreme thermophile Thermococcus kodakaraensis KOD1, possess- es superior processivity and fidelity that enable faster, more accurate PCR amplification than with conventional KOD DNA Polymerase enzymes, including Pfu DNA polymerase (1). KOD HiFi DNA Polymerase is also available in a Hot Start version Faster than Taq... for high specificity and increased read length (2), and as a blend (KOD XL DNA Polymerase) recommended for very long templates (3). More accurate than Pfu...

NovaTaq™ DNA Polymerase is a high-purity, recombi- nant enzyme suitable for any application requiring premi- um quality Taq DNA polymerase. For increased specifici- ty and convenience with standard PCR, we also offer NovaTaq™ Hot Start DNA Polymerase and the Taq Antibody.NovaTaq Hot Start DNA Polymerase is a chem- ically modified form of Taq DNA polymerase that 1. Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., becomes active when heated at 95°C for 7–10 minutes. Kawakami, B., Oka, M., and Imanaka, T. (1997) Appl. Environ. Microbiol. 63, The Taq Antibody is available as an alternative means to 4504–4510. provide hot start capability to NovaTaq DNA Polymerase 2. Mizuguchi, H., Nakatsuji, M., Fujiwara, S., Takagi, M., and Imanaka, T. (1999) J. Biochem. (Tokyo) 126, 762–768. as well as other sources of Taq DNA polymerase. Please 3. Nishioka, M., Mizuguchi, H., Fujiwara, S., Komatsubara, S., Kitabayashi, M., refer to the table above as a guide to select the appro- Uemura, H., Takagi, M., and Imanaka, T. (2001) J. Biotechnol. 88, 141–149. priate enzyme combination for your application.

3 T ABLE O F C ONTENTS

A MPLIFY (see page 6)

KOD POLYMERASES ■ KOD DNA Polymerase is a new, high performance DNA polymerase

KOD HiFi DNA Polymerase – Faster than Taq,more accurate than Pfu KOD Hot Start DNA Polymerase – A heat activatable form of KOD HiFi DNA Polymerase for automated PCR set-up KOD XL DNA Polymerase – Recombinant forms of KOD Polymerase blended for long and accurate PCR

GENOMIC DNA Genomic DNA – High quality, high integrity human, animal, and yeast DNA

RT-PCR TOOLS First Strand cDNA Synthesis Kit – Reliable preparation of templates for RT-PCR One Step RT-PCR Kit – Convenient, one-enzyme, single buffer system for RT-PCR

TAQ POLYMERASES AND PCR KITS ■ NovaTaq™ DNA Polymerase is a premium quality recombinant form of Thermus aquaticus DNA polymerase

NovaTaq DNA Polymerase – A molecular biology lab essential 10 mM dNTP Mix NovaTaq PCR Kit – Everything for PCR except DNA and primers NovaTaq PCR Kit PLUS – What you need for successful PCR and PCR optimization NovaTaq PCR Master Mix – A ready-to-use 2X concentrated PCR reagent mixture NovaTaq Hot Start DNA Polymerase – For increased specificity and convenient reaction assembly Taq Antibody – Monoclonal antibody for automated Hot Start PCR

O RDERING I NFORMATION (see page 24)

4 www.novagen.com P URIFY (see page 14)

DNA PURIFICATION SpinPrep™ PCR Clean-up Kit – Rapid purification of PCR products for downstream procedures SpinPrep Gel DNA Kit – Rapid, efficient extraction of DNA from agarose gels SpinPrep Plasmid Kit – Rapid, high quality plasmid minipreps SpinPrep Master Kit – Combo Kit; 20 plasmid preps and 20 DNA gel extractions

PELLET PAINT® CO-PRECIPITANTS Pellet Paint Co-Precipitant – Rapid, quantitative precipitation of DNA and RNA, including PCR clean-up Pellet Paint NF Co-Precipitant – Non-fluorescent visible DNA co-precipitant for sequencing and other automated applications

C LONE (see page 18)

CLONING KITS Perfectly Blunt® Cloning Kits – Efficient “universal” cloning of DNA amplified by any polymerase AccepTor™ Vector Kits – Rapid, direct cloning of DNA amplified with non-proofreading DNA polymerases

T RANSFORM (see page 20)

COMPETENT CELLS NovaBlue Singles™ Competent Cells – High efficiency transformation of E. coli in less than 8 minutes HT96™ NovaBlue Competent Cells – High-efficiency competent cells pre-dispensed in a 96-well format HT96 Isothermal Block – Efficient thermal transfer for uniform incubations

A NALYZE (see page 22)

DNA LADDERS AND MARKERS Perfect DNA™ Ladders and Markers PCR Markers

5 KOD POLYMERASES

Pure recombinant high fidelity DNA polymerases from Thermococcus kodakaraensis

KOD HiFi DNA Polymerase Ultra high fidelity DNA polymerase

KOD HiFi DNA Polymerase is a recombinant form of Thermococcus ADVANTAGES kodakaraensis KOD1 DNA polymerase. It is the most efficient •Higher fidelity than Pfu DNA Polymerase (1) thermostable DNA polymerase, exhibiting higher accuracy, elongation

rate and processivity than any other commercially available DNA poly- •Higher processivity – sequential nucleotide polymerization merase. KOD HiFi DNA Polymerase possesses 3' ← 5' is between 10 and15X greater than Pfu DNA Polymerase and exonuclease-dependent proofreading activity that enables the Deep Vent* DNA Polymerase (1) polymerase to correct nucleotide misincorporation. The enzyme •Greater yield – extension speed is 2X faster than Taq DNA generates blunt-ended PCR fragments that are suitable for cloning polymerase and 5X faster than Pfu DNA polymerase (1) with Novagen’s Perfectly Blunt® Cloning Kits. • More accurate PCR in a shorter time Each order also includes 10X KOD HiFi DNA Polymerase Buffer 1 and 10X KOD HiFi DNA Polymerase Buffer 2, plus separate vials of 25 mM •No truncated amplification products in PCR reaction

MgCl2 and 2mM dNTP Mix.

123 4 KOD HiFi Pfu Taq bp 1 Perfect DNA™ Markers, 0.5–12 kbp Enzyme DNA Polymerase DNA Polymerase DNA Polymerase 12,000 – 2 5.4 kbp PCR product (lambda DNA) 10,000 – Species Thermococcus Pyrococcus Thermus 8000 – 3 2.0 kbp PCR product (plasmid DNA) 6000 – kodakaraensis furiosis aquaticus YT-1 41.6 kbp PCR product (human genomic DNA) 4000 – † –3 –3 –2 Fidelity 3.5 x 10 3.9 x 10 1.3 x 10 3000 –

Elongation rate 106–138 25 61 2000 – (bases/second) 1500 – Processivity > 300 bases < 20 bases not determined (nucleotide bases) 1000 –

1. Takagi, M., et al., (1997) Applied and Environmental Microbiology 63, 4504–4510. PCR products amplified using KOD HiFi DNA Polymerase † Fidelity was measured by the authors as mutation frequency in PCR products using DNA fragments from various templates were amplified using 2.5 a sensitive blue/white phenotypic assay with a 5.2 kbp lacZ plasmid as template. units KOD HiFi DNA Polymerase in a standard 100 µl reaction. * Deep Vent is a trademark of New England Biolabs, Inc. Portions of each amplification reaction were analyzed by agarose gel electrophoresis (1.2% TAE).

6 www.novagen.com AMPLIFY KOD Hot Start DNA Polymerase Heat-activatable form of KOD HiFi DNA Polymerase for automated PCR set-up

KOD Hot Start DNA Polymerase is a premixed complex of KOD HiFi ADVANTAGES

DNA Polymerase and two monoclonal antibodies that inhibit the ← •Highest accuracy, yield and processivity among enzyme’s DNA polymerase and 3' 5' exonuclease activities during PCR commercially available proofreading DNA polymerases (1) assembly. KOD Hot Start DNA Polymerase combines the high fidelity, fast extension speed, and outstanding processivity of KOD HiFi DNA • Amplifies genomic DNA templates up to 12 kbp Polymerase with the high yield and specificity of the antibody-based Hot • Amplifies plasmid DNA templates up to 21 kbp Start technology. KOD Hot Start DNA Polymerase generates blunt-ended PCR fragments that are suitable for cloning with Novagen’s Perfectly • Successfully amplifies GC-rich sequences Blunt® Cloning Kits. • Eliminates mispriming and primer-dimer formation

Each order also includes 10X KOD Hot Start DNA Polymerase Buffer • Convenient room temperature set-up plus separate vials of 25 mM MgSO4 and 2mM dNTP Mix.

M 8.4 12.3 (kbp) M 1 2 4 6 8 10 12 15 21 M (kbp)

Genomic DNA amplification The human myosin heavy chain Lambda DNA amplification gene (8.4 kbp) and human ß-globin The indicated fragments of gene (12.3 kbp) were amplified lambda DNA were amplified using KOD Hot Start DNA with KOD Hot Start DNA Polymerase Polymerase

M=Markers M=Markers PCR h i g h f i d e l i t y

For ordering information, see page 24. KOD POLYMERASES

KOD XL DNA Polymerase High performance enzyme blend for long and accurate PCR

KOD XL DNA Polymerase is an optimized blend of KOD HiFi DNA ADVANTAGES Polymerase and a mutant form of KOD HiFi that is deficient in 3' 5'← •Ideal for amplification of large DNA fragments exonuclease activity. The enzyme mixture is designed for reliable from purified DNA or crude samples amplification of long, complex targets with robust yield and high accuracy. KOD XL DNA Polymerase generates PCR products suitable • Amplifies DNA templates up to 30 kbp for cloning with Novagen’s AccepTor™ Vector and Perfectly Blunt® • Successfully amplifies GC-rich sequences Cloning Kits. •Efficiently incorporates derivatized dNTPs Each order also includes 10X KOD XL DNA Polymerase Buffer and 2 mM dNTP Mix.

Performance comparison λ DNA fragments were amplified using Taq DNA Polymerase and KOD XL DNA Polymerase

M λHind III DNA Markers 58 kbp PCR product 11 kbp PCR product 6 10 kbp PCR product 22 kbp PCR product 7 12 kbp PCR product 34 kbp PCR product 8 15 kbp PCR product 46 kbp PCR product

Genomic DNA High molecular weight, high purity DNA for any application ORGANISM SOURCE Bovine blood Novagen’s genomic DNAs are qualified for genomic analysis including Cat blood PCR and library construction. At least 90% of the DNA is greater than Chicken blood 100 kbp in size, as analyzed by CHEF gel electrophoresis (> 50 kbp for Dog blood Saccharomyces cerevisiae). No inhibition of restriction enzyme activity is Human (female only) blood observed, and the DNA is free of contaminating RNA and protein. Human (male only) blood Human (male & female) blood

ladder dog chicken bovine pig Mouse blood bovine pig λ kbp M U E S U E S U E S U E S kbp Pig blood – 468 Rat (Sprague Dawley) blood 23 – 0.7% agarose gel showing digestion of genomic S. cerevisiae (wild type S288C) mixed culture – 195 9.4 – DNA (5 µg/lane, gel 6.7 – stained with ethidium bromide). All DNAs are supplied at 100–300 µg/ml in TE buffer, 2.3 – 100 µg per vial. – 39 2.0 – U = no enzyme E = EcoR I S = Sau3A I 8 CHEF analysis Restriction enzyme analysis long AMPLIFY RT-PCR TOOLS

First Strand cDNA Synthesis Kit Reliable preparation of templates for RT-PCR

The First Strand cDNA Synthesis Kit is designed for the preparation of high quality first strand cDNA from cellular RNA templates.The kit contains MMLV Reverse Transcriptase for superior yields of full-length cDNA. Both oligo(dT) and random hexamer primers are included for a choice of general priming strategies as alternatives to user-supplied specific primers. The reaction conditions are compatible with direct

addition of primers and KOD Hot Start DNA Polymerase or KOD XL M AS AS + RH dT AS + dT RH DNA Polymerase for amplification of the cDNA product. PCR of first strand cDNA The Positive Control RNA was subjected to ADVANTAGES first strand cDNA synthesis with various primer combinations followed by addition of the 5' • MMLV Reverse Transcriptase provides superior yields of sense Control Primer (and antisense Control full-length cDNA Primer, where appropriate) and amplification. First strand primers are indicated. • Both oligo(dT) and random hexamer primed synthesis options AS = antisense Control Primer, RH = random hexamers, dT = oligo(dT) •Kit configured for direct addition of reagents and primers

• Positive Control RNA and Control Primers included in kit

One Step RT-PCR Kit Convenient one-enzyme, single buffer system for RT-PCR

The One Step RT-PCR Kit is designed for rapid, sensitive detection of Each kit provides sufficient reagents to perform 50 RT-PCR reactions. gene expression in tissues and cells. The kit takes advantage of the Positive Control RNA and Control Primers are also included. properties of recombinant Thermus thermophilus (rTth) DNA ADVANTAGES Polymerase, which acts as both a thermostable RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase. In a single • Robust one-step, one-enzyme system for easy reaction, cDNA is synthesized from input RNA through reverse reaction assembly transcription, followed by PCR amplification of the cDNA without • Eliminates the risk of cross-contamination associated with two changing the buffer or adding reagents.The amplified DNA is normal- step RT-PCR protocols ly analyzed by agarose gel electrophoresis. •High temperature (60°C) reverse transcription enhances Use of gene specific primers or oligo(dT) and one gene specific 5' read-through of RNA secondary structure or high primer is recommended with the kit. Amplification of a specific GC content message requires approximately two hours. •Ideal for gene expression studies

ACCURATE For ordering information, see page 24. TAQ P OLYMERASES AND PCR KITS

NovaTa q™ DNA Polymerase bp 1234567 1 Perfect DNA™ Markers, 0.5–12 kbp A premium quality recombinant form of 12,000 – 8000 – 2 0.5 kbp PCR product Thermus aquaticus DNA polymerase 6000 – 4000 – 3 1.0 kbp PCR product 3000 – 4 2.0 kbp PCR product NovaTaq™ DNA Polymerase is suitable for a wide range of PCR 2000 – 5 4.8 kbp PCR product applications. Each preparation is quality tested to ensure the highest 1500 – 67.35 kbp PCR product purity and reproducible performance. The enzyme leaves single 7 Perfect DNA Markers, 0.5–12 kbp 3'-dA overhangs that make the products suitable for cloning with 1000 – Novagen’s AccepTor™ Vector and Perfectly Blunt® Cloning Kits.

Each order also includes optimized 10X NovaTaq Buffer containing 500 –

MgCl2 for routine amplification conditions, plus separate vials of 10X NovaTaq Buffer without MgCl and 25 mM MgCl to enable 2 2 PCR products amplified using NovaTaq DNA Polymerase 2+ convenient optimization of Mg concentrations. DNA fragments 0.5 kbp to 7.35 kbp in size were amplified using 2.5 units NovaTaq DNA Polymerase in a standard 100 µl reac- tion. Products from each amplification reaction were analyzed by agarose gel electrophoresis (1.2% TAE). 10 mM dNTP Mix

This dNTP Mix is a ready-to-use preparation of ultrapure dATP, dCTP, dGTP, and dTTP (monosodium salts) at a concentration of 10 mM each in sterile deionized water at pH 7.0.The dNTP Mix is free of RNase and DNase and is qualified for any application that requires pure deoxynucleotides, such as PCR, cDNA synthesis, and fill-in reactions.

NovaTa q PCR Kit

The NovaTaq PCR Kit includes all the reagents necessary for PCR amplification except primers and template. Each component of the kit has been quality tested in PCR applications. Sufficient amounts of reagents are provided for 200 standard 50 µl (or 100 × 100 µl) ampli- fication reactions.

The kit includes NovaTaq DNA Polymerase (250 U at 5 U/µl), 10X

NovaTaq Buffer with MgCl2, 10X NovaTaq Buffer without MgCl2,25

mM MgCl2, 10 mM dNTP Mix and PCR Grade Water.

10 www.novagen.com AMPLIFY NovaTa q™ PCR Kit PLUS

The NovaTaq™ PCR Kit PLUS includes 1.5 ml of 10X NovaTaq Optimization Buffer in addition to all of the NovaTaq PCR Kit reagents. The NovaTaq Optimization Buffer offers advantages of greater tolerance to variable Mg2+ concentrations and a wider temperature window for optimal primer: template annealing. Sufficient amounts of reagents are provided for 200 standard 50 µl amplification reactions (or 100 × 100 µl reactions).

The kit includes NovaTaq DNA Polymerase (250 U at 5 U/µl), 10X

NovaTaq Buffer with MgCl2,10X NovaTaq Buffer without MgCl2, 10X

NovaTaq Optimization Buffer, 25 mM MgCl2, 10 mM dNTP Mix and PCR Grade Water.

ADVANTAGES

• Maximum PCR reaction versatility and optimization

•PCR optimization buffer included

NovaTa q PCR Master Mix

NovaTaq™ PCR Master Mix is a ready-to-use 2X concentrated mixture of NovaTaq DNA Polymerase, ultrapure deoxynucleotides, and reaction buffer without MgCl2. The Master Mix simplifies the assembly of PCR reactions and offers advantages of time savings, consistency, and minimal risk of con- tamination. Simply add the NovaTaq PCR Master Mix to an equal volume containing the required amount of MgCl2, DNA template, and primers, and the reaction is ready for thermal cycling.The final diluted reaction contains 2.5 U of NovaTaq DNA Polymerase per 100 µl. Sufficient components are included for 100 amplification reactions.

The kit includes NovaTaq PCR Master Mix, 25 mM MgCl2 and PCR Grade Water.

ADVANTAGES

• Reproducibility

•Accuracy; no pipetting errors •Minimal risk of contamination quality • Convenience performance

For ordering information, see page 25. TAQ P OLYMERASES AND PCR KITS

NovaTa q™ Hot Start DNA Polymerase Heat-activatable modified form of recombinant Taq DNA Polymerase

NovaTaq™ Hot Start DNA Polymerase is a heat-activatable, chemical- ADVANTAGES ly modified form of Taq DNA polymerase that is inactive at room tem- •Higher PCR specificity and yield perature. NovaTaq Hot Start DNA Polymerase provides improved specificity when compared to standard Taq DNA polymerase and can •Improved low-copy target amplification eliminate generation of non-specific amplification products such as • Automated room temperature set up primer-dimers and misprimed products.The enzyme must be activat- ed by heat treatment (7–10 min at 95˚C) before polymerization is •Target amplification of up to 5 kbp possible. The enzyme leaves single 3'-dA overhangs that make the •Ideal for quantitative PCR applications products suitable for cloning with Novagen’s AccepTor™ Vector Kits.

Each order also includes optimized 10X NovaTaq Hot Start Buffer and

a separate vial of 25 mM MgCl2.

Activation Profile of NovaTaq Hot Start DNA Polymerase

1 2 3

Hot Start PCR products

1Perfect DNA™ Markers, 0.05–10 kbp 21.0 kbp DNA fragment amplified using NovaTaq Hot Start DNA Polymerase 31.0 kbp DNA fragment amplified using Competitor A chemically-modified Taq DNA polymerase

12 www.novagen.com AMPLIFY Ta q Antibody Converts unmodified Taq DNA polymerase into a hot start enzyme

The Taq Antibody is a mouse monoclonal antibody that inhibits Taq DNA polymerase activity at ambient temperatures. It provides an anti- body-mediated hot start that enhances the specificity, sensitivity, and convenience of PCR. Inhibition is effective during reaction assembly at room temperature, and is completely reversed when thermal cycling begins, with no other effect on PCR conditions.The antibody inhibits both native and recombinant Taq DNA polymerase activities, including NovaTaq DNA Polymerase.

One microgram (1 µl) of antibody inhibits > 95% of 5 units of Taq DNA polymerase. For convenience, simply mix 100 µl Taq Antibody with 500 U (100 µl) NovaTaq DNA Polymerase, incubate for 5 minutes at room temperature, and proceed with PCR. The polymerase:anti- body complex can be freshly prepared for each experiment or stored at –20°C for later use.

ADVANTAGES

•Higher PCR specificity and yield

•Improved low-copy target amplification

• Room temperature set-up compatible with automation

high throughput HEAT ACTIVATABLE

For ordering information, see page 25. DNA PURIFICATION

SpinPrep™ DNA Purification Kits

The SpinPrep™ Kits are silica membrane-based DNA purification format. Protocols do not require organic extraction or precipitation, systems that are easy to use and yield pure DNA quickly. For routine and the DNA is eluted in TE buffer, ready for sequencing, subcloning, clean-up of PCR products, isolation of DNA from gels, and purification labeling, transformation, or PCR.The SpinPrep Master Kit combines of high quality plasmid DNA, these kits offer a convenient spin column materials from the SpinPrep Plasmid Kit and SpinPrep Gel DNA Kit.

SpinPrep SpinPrep SpinPrep PCR Clean-up Kit Gel DNA Kit Plasmid Kit Rapid purification of PCR products for Rapid, efficient extraction of DNA from Rapid, high quality plasmid minipreps

downstream procedures agarose gels 1 ml culture 3 ml culture high copy plasmid low copy plasmid 12 3 45 12 3 456 bp 123 4 bp

12,000 – 2000 – 1500 – 8000 – 1000 – 18 750 – 16 1000 – 500 – 14 300 – 12 150 – 150 – 10 8 50 – 6

µg plasmid eluted 4 Lane Sample Recovery 2 1PCR Markers 1control mix – 0 2 crude PCR product 2 150 bp 90% 123456 gel lane 3 purified PCR product 31,000 bp 90% 4PCR Markers 48,000 bp 50% 5 12,000 bp 52%

Primer removal with the SpinPrep PCR Gel analysis and yield of DNA fragments Gel analysis and yield of plasmids isolated Clean-up Kit isolated with the SpinPrep Gel DNA Kit with the SpinPrep Plasmid Kit

SpinPrep Master Kit Materials provided for 20 plasmid minipreps and 20 extractions from agarose gels

14 www.novagen.com PURIFY Product Scale DNA Yield Time Required Size Range Recommended Applications Procedure SpinPrep™ PCR DNA in PCR Membrane binding < 10 minutes 100 bp to Ligation, labeling, sequencing, •Layer sample on membrane. Spin. Clean-up Kit reactions capacity 6 µg > 12,000 bp PCR, transformation, •Add wash buffer. Spin. (100 µl per rxn) in vitro transcription •Add elution buffer. Spin.

SpinPrep DNA in agarose Membrane binding < 20 minutes 150 bp to Ligation, labeling, sequencing, •Dissolve gel slice (in chaotrope provided) Gel DNA Kit gel slices capacity 20 µg > 12,000 bp PCR, transformation, •Transfer to membrane. Spin. (150 mg per rxn) in vitro transcription •Add wash buffer. Spin. • Add elution buffer. Spin.

SpinPrep 1–3 ml culture Up to 20 µg < 30 minutes up to 20 kbp Sequencing, restriction digest, •Alkaline lysis of E. coli (3 steps) Plasmid Kit transformation •Transfer supernatant to membrane. Spin. • Add wash buffer. Spin. •Add elution buffer. Spin.

ADVANTAGES

• Fast and easy

•High yield

•Proven silica membrane technology

•Simple, reproducible procedure

speed Purity For ordering information, see page 26. P ELLET P AINT® C O -PRECIPITANTS

Pellet Paint Co-Precipitant

Rapid, quantitative precipitation of DNA and RNA, including PCR clean-up

Pellet Paint® Co-Precipitant* is a visible dye-labeled carrier formulated specifically for use in alcohol precipitation of nucleic acids. The five-minute precipitation protocol requires no low temperature ADVANTAGES incubations or prolonged centrifugation. Both RNA and DNA are efficiently precipitated from solutions as dilute as 2 ng/ml, and the • Visibility maximizes sample recovery pellet is easily located by its vivid pink color.The pellet can be easily •Suitable for DNA and RNA precipitation followed during washing steps, preventing losses during handling. Pellet Paint is compatible with most molecular biology procedures and is free • Compatible with ethanol and isopropanol precipitation of contaminating nucleic acids and nucleolytic enzymes. Pellet Paint is • Compatible with sodium acetate, sodium chloride and compatible with Cy5-based automated sequencers. Pellet Paint NF ammonium acetate salts (below) is recommended for use with PE Applied Biosystems • Room temperature incubation and short centrifugation times automated sequencers. •Preparations free of contaminating nucleic acids and protein

•Improves yield, reproducibility, and sensitivity in many applications (e.g. PCR)

•Works even with minute quantities of nucleic acid (< 2 ng/ml)

Pellet Paint NF Co-Precipitant •Compatible with numerous downstream applications Non-fluorescent visible co-precipitant for automated sequencing applications •Pellet Paint NF Co-Precipitant compatible with BigDye Pellet Paint NF Co-Precipitant* is a non-fluorescent dye-labeled terminators and other fluorescent applications carrier compatible with fluorescent sequencing and other applications. It can increase precipitation speed and efficiency, lower centrifugation times, and help rapidly clean up unincorporated BigDye™ terminators. Since nucleic acid pellets with Pellet Paint NF Co-Precipitant are so visible, small pellets are unlikely to be lost and pellet dissolution is clearly visible.

Pellet Paint NF Co-Precipitant is fully compatible with the ABI PRISM® BigDye Terminator Cycle Sequencing Ready Reaction.To avoid extra sample handling, Pellet Paint NF Co-Precipitant can be added directly to the reaction mix, template DNA, crude PCR samples, or dilution buffer prior to the cycle sequencing reaction. Pellet Paint NF Co- Precipitant has no detectable effect on the sequencing reaction or *patent pending BigDye and ABI PRISM are trademarks of the PE Corporation sequencing accuracy.

16 www.novagen.com PURIFY PELLET PAINT® PROCEDURE

1 Add 2 µl Pellet Paint or Pellet Paint NF Co-Precipitant + 0.1 volume 3 M Na Acetate to sample and mix briefly 2

Add 2 volumes ethanol (or 1 volume isopropanol) and briefly vortex 3

Incubate at room temperature for 2 minutes 4

Spin sample for 5 minutes 5

Discard supernatant, wash and resuspend pellet

USE IT so you don’t lose it

For ordering information, see page 26. C LONING K ITS

Less than 1 hour from PCR product to plating

Perfectly Blunt® Cloning Kits Simplified cloning of DNA generated by PCR using any type of DNA polymerase

The Perfectly Blunt® Cloning Kits are designed for simplified cloning of Six different plasmid vectors are available in Perfectly Blunt Cloning Kits. DNA generated by PCR using any type of DNA polymerase. This Vector choices include those designed for general cloning, sequencing, approach enables the use of high fidelity proofreading enzymes for optimal transcription/translation, and optimal protein expression in E. coli. amplification, thus decreasing the probability of generating mutations in Please refer to the table on the facing page for features and applications of the target sequence. With the Perfectly Blunt cloning protocol, you can the vectors. go from PCR product to plating transformants in less than 1 hour with minimal hands-on time.

The finished PCR reaction product is converted to a blunt, phosphorylat- ADVANTAGES ed form in a 15-minute reaction using premixed reagents. Following a 5- •Blunt methodology is 3- to 24-fold more efficient than T-vector minute heat inactivation step, the treated insert is combined with the cloning methods (1) ready-to-use vector and ligated in an optimized reaction using premixed • No restriction enzymes or special primers reagents. An exclusive 8-minute transformation procedure using highly •Direct ligation of PCR product with vector efficient NovaBlue Singles™ Competent Cells generates recombinant •Compatible with any DNA polymerase colonies that are easily visualized by blue/white screening. • Blue/white screening with all vectors •Simple protocol takes as little as 45 minutes from PCR product to plating transformants 1. Novy, R.E., Yaeger, K.W. and Kolb, K.M. (1996) inNovations 6, 7–11

P

Z la lac cZ PCR product P r ve cto cto AccepTor™ Vector Kits* ve r Rapid, direct cloning of DNA amplified with non-proofreading DNA polymerases

The AccepTor™ Vector Kits are designed for simplified cloning of ADVANTAGES PCR products generated using non-proofreading thermostable DNA • No restriction enzymes or special primers polymerases, such as native and recombinant Taq polymerase, that leave single 3'-dA overhangs on the reaction products.The linearized •Compatible with polymerases that leave single 3'-dA overhangs AccepTor Vector contains single 3'-dU DNA ends that are •Simple protocol takes less than 40 minutes from PCR product to compatible with direct ligation of these products without the need for plating transformants intermediate reactions.The dU residues are converted to dT residues • Blue/white screening with pSTBlue-1 and pETBlue™-1 vectors in vivo following transformation.

*AccepTor Vector Kits are covered under U.S. Patent No. 5,856,144 issued to Novagen, Inc.

dA dU A cZ d dU la la PCR product cZ r ve cto cto 18 www.novagen.com ve r CLONE Perfectly Blunt® Vectors

Vectors Applications Vector Advantages pSTBlue-1 Archiving, Subcloning, Dual opposed SP6/T7 promoters Sequencing, Amp or Kan selection In vitro transcription Dual EcoR I sites flank insert pT7Blue-3 T7 promoter Amp or Kan selection Dual EcoR I sites flank insert pT7Blue T7 promoter Nde I/BamHI sites flank insert pT7Blue-2 Protein expression: T7-driven in vitro protein synthesis In vitro transcription/translation, N-terminal S•Tag™ sequence Sequencing Optimal Kozak translation initiation Xenopus globin 5' UTR pETBlue-1 Protein expression: T7lac-driven No fusion tags tightly controlled, high level Insert provides ATG start codon expression in E. coli pETBlue-2 Optional C-terminal HSV•Tag®, His•Tag® Vector provides ATG start codon

AccepTor™ Vectors

Vectors Applications Vector Advantages

pSTBlue-1 Archiving, Subcloning, Dual opposed SP6/T7 promoters Sequencing, Amp or Kan selection In vitro transcription Dual EcoR I sites flank insert pETBlue™-1 Protein expression: No fusion tags T7lac-driven, tightly Insert provides ATG start codon controlled, high level expression in E. coli

F AST simple

For ordering information, see page 26 C OMPETENT C ELLS

NovaBlue Singles™ Competent Cells Premeasured, ready-to-use aliquots for speedy, hassle-free transformation

NovaBlue is an E. coli K-12 strain ideally suited as an initial cloning host Plasmid and SOC Medium for the specified number of transformation due to its high transformation efficiency, blue/white screening reactions. capability, and recA endA mutations, which result in high yields of excel- ADVANTAGES lent quality plasmid DNA. The NovaBlue Singles™ Competent Cells format is designed for ultimate convenience and reliability in plasmid • Convenient, one tube per transformation. No transformation. Cells are provided in single use aliquots that eliminate measuring, subdividing, shortage, waste or ß-ME the need to subaliquot, freeze/thaw or waste partially used vials, thus •Simple, reliable saving time and increasing performance.To use, simply thaw, add DNA, incubate 5 minutes on ice, heat shock for 30 seconds, place on ice for • Cost-effective without leftovers 2 minutes and plate directly (when selecting for ampicillin resistance) • Consistently high transformation efficiency or after incubation at 37°C for 30 minutes (when selecting for •Transformation in as little as 8 minutes kanamycin resistance). • Easy-open flip caps NovaBlue Singles Competent Cells are provided as frozen 50 µl aliquots, each intended for one transformation, and also include Test

HT96™ NovaBlue Competent Cells High efficiency competent cells pre-dispensed in a 96-well format

The HT96™ NovaBlue Competent Cells are designed for high throughput cloning. The Cells are pre-dispensed in a robust 96-well ADVANTAGES polypropylene plate, which is compatible with a variety of thermal cyclers as well as water baths for performing the transformation reac- • Seal may be pierced or peeled off tion.The wells are individually sealed and have raised rims to prevent •Cap strips provided for resealing cross-contamination. The seal may be easily pierced with standard pipet tips or peeled off for access. Strips of caps are also •Plate may be separated into smaller groups of wells provided for reliable sealing during manipulation and storage. Groups •Compatible with many thermal cyclers of 24 wells can by simply detached from the whole plate for process- ing smaller numbers of samples.

HT96 NovaBlue Competent Cells are provided as 20 µl aliquots in a 96-well plate. Each package also includes cap strips, SOC Medium,Test Plasmid and a Reagent Reservoir.

20 www.novagen.com TRANSFORM HT96™ Isothermal Block Efficient thermal transfer for uniform incubations

The HT96™ Isothermal block is an anodized aluminum, solvent-resistant block specially designed to hold HT96 plates. It provides efficient thermal transfer during the low temperature incubation and heat-shock steps used in transformation protocols. Simply pre-incubate the anodized alu- minum block at the desired temperature and place the HT96 Competent Cell plate in the block. The overall block dimensions are 12 cm x 7.6 cm x 2.5 cm and it is compatible with most 96 well PCR plates. The HT96 plate is shown in the HT96 Isothermal Block in the photo below.

High throughput cloning using HT96 Competent Cells

Blue/white screening with NovaBlue Competent Cells

That’s it! 3 Steps 15 Minutes

For ordering information, see page 27. DNA LADDERS AND M ARKERS

bp Perfect DNA Ladders Perfect DNA™ Ladders and Markers – 3000

– 2000 Perfect DNA™ Ladders and Markers are available in several conven- ient size ranges for gel analysis.The DNA species are supplied in equal

masses, except for a 2–3X reference band in some marker sets. Each bp bp marker or ladder is supplied in 1X DNA Gel Loading Buffer. A – 1000 – 900 separate vial of 6X DNA Gel Loading Buffer is included. – 800 – 1000 – 700 – 10,000 – 900 – 8000 – 600 – 800 – 6000 – 700 – 5000 ADVANTAGES – 500 – 4000 – 600 – 450 – 3000 – 400 – 500 – 2500 •Ready-to-use mixtures – 350 – 2000 – 400 – 300 – 1500 •Evenly spaced, easy-to-remember sizes – 250 – 300 – 1000 – 200 • Includes gel loading buffer – 150 – 200 – 500 – 100 • Consistent lot-to-lot, quality-assured product ensures – 100 – 50 reproducible results 50 bp Ladder 100 bp Ladder 1 kbp Ladder •Available in various size ranges 2.0% TAE agarose gel 2.0% TAE agarose gel 0.8% TAE agarose gel

Perfect DNA Markers bp – 10,000 – 8000 – 6000 – 4000 – 3000 bp – 12,000 – 10,000 – 2000 – 8000 bp – 6000 – 1500 – 1400 – 12,000 – 10,000 – 4000* – 8000 – 1000 – 6000 – 3000

– 750 – 4000* – 2000 – 3000 – 1500 – 500

– 2000 – 400 – 1000 – 1000 – 300

– 1500 – 500* – 200 – 400 – 300 – 100 – 200 – 500 – 100 – 50 0.5–12 kbp 0.1–12 kbp 0.05–10 kbp 0.8% TAE agarose gel 1.5% TAE agarose gel 2.0% TAE agarose gel 22 www.novagen.com ANALYZE PCR Markers

The PCR Markers are a mixture of eight defined DNA fragments for characterizing small DNA products. The markers are supplied in gel loading buffer containing two tracking dyes that do not interfere with UV illumination of ethidium bromide-stained bands. A separate vial of 6X Loading Buffer is included. The recommended amount for loading produces bands of even intensity that are bright, sharp, and easy to photograph.

ADVANTAGES

•Ready-to-use mixture

•Accurate sizing of PCR poducts

• Includes gel loading buffer

• Consistent lot-to-lot, quality-assured product ensures reproducible results

bp – 2000

– 1500

– 1000

– 750

– 500

– 300

– 150

– 50

50–2000 bp 1.5% TAE agarose gel wide range perfectly accurate For ordering information, see page 27. O RDERING I NFORMATION

PRODUCT SIZE CAT NUMBER PRICE

KOD HIFI DNA POLYMERASE * 250 U 71085-3 £148 COMPONENTS: • 250 U KOD HiFi DNA Polymerase (2.5 U/µl ) •1 ml 10X KOD HiFi DNA Polymerase PCR Buffer #1 •1 ml 10X KOD HiFi DNA Polymerase PCR Buffer #2 •1 ml 2 mM dNTP Mix

•1 ml 25 mM MgCl 2

KOD HOT START DNA POLYMERASE * 200 U 71086-3 £128 5 × 200 U 71086-4 £480 COMPONENTS: • 200 U or 5 × 200 U KOD Hot Start DNA Polymerase (2.5 U/µl) • 1.2 ml or 5 × 1.2 ml 10X KOD Hot Start PCR Buffer

•1 ml 25 mM MgSO4 •1 ml or 5 × 1 ml 2mM dNTP Mix

KOD XL DNA POLYMERASE * 250 U 71087-3 £165 5 × 250 U 71087-4 £640 COMPONENTS: • 250 U or 5 × 250 U KOD HiFi DNA Polymerase (2.5 U/µl ) •1 ml or 5 × 1 ml 10X KOD XL DNA Polymerase PCR Buffer •1 ml or 5 × 1 ml 2 mM dNTP Mix

GENOMIC DNA Bovine blood 69231-3 £80 Cat blood 69235-3 £80 Chicken blood 69233-3 £80 Dog blood 69234-3 £80 Human (female only) blood 70605-3 £80 Human (male only) blood 70572-3 £80 Human (male & female) blood 69237-3 £80 Mouse blood 69239-3 £80 Pig blood 69230-3 £80 Rat (Sprague Dawley) blood 69238-3 £80 S. cerevisiae (wild type S288C) mixed culture 69240-3 £80 All DNAs are supplied at 100–300 µg/ml in TE buffer, 100 µg per vial.

FIRST STRAND CDNA SYNTHESIS KIT 40 rxn 69001-3 £159 COMPONENTS:•20 µg Oligo(dT) Primer • 4000 U MMLV Reverse Transcriptase • 10 µg Random Hexamer Primers •0.2 ml 5X First Strand Buffer • 1.5 ml Nuclease-free Water •0.1 ml 100 mM DTT • Positive Control RNA • 50 µl 10 mM dNTP Mix • Positive Control Primers

ONE STEP RT-PCR KIT * 50 rxn 71089-3 £176 COMPONENTS: • 250 U rTth DNA Polymerase (2.5 U/µl) • 2 × 1.1 ml RNase Free Water •1 ml 5X Reaction Buffer • 0.05 ml 10 mM G3PDH Control Primer F •0.5 ml 25 mM Mn(OAc) •0.05 ml 10 mM G3PDH Control Primer R 24 2 •0.3 ml 2.5 mM dNTP Mix • 0.05 ml Positive control Human G3PDH •0.1 ml RNase Inhibitor (10 U/µl) RNA (5 x 108 copies/ml) Prices subject to change PRODUCT SIZE CAT NUMBER PRICE

NOVATAQ™ DNA POLYMERASE 100 U 71003-3 £38 500 U 71003-4 £165 2,500 U 71003-5 £759 ADDITIONAL COMPONENTS: × •1 or 2 or 7 1.5 ml 10X NovaTaq Buffer with MgCl2 × •1 or 2 or 7 1.5 ml 10X NovaTaq Buffer without MgCl2 × •1 or 2 or 7 1.5 ml 25 mM MgCl2

10 MM DNTP MIX 0.2 ml 71004-3 £33

NOVATAQ PCR KIT 250 U 71005-3 £119 COMPONENTS:

• 250 U NovaTaq DNA Polymerase (5 U/µl) • 1.5 ml 25 mM MgCl2

•1.5 ml 10X NovaTaq Buffer with MgCl2 •0.2 ml 10 mM dNTP Mix × •1.5 ml 10X NovaTaq Buffer without MgCl2 •5 2ml PCR Grade Water

NOVATAQ PCR KIT PLUS 250 U 71006-3 £127 COMPONENTS:

• 250 U NovaTaq DNA Polymerase (5 U/µl) • 1.5 ml 25 mM MgCl2

•1.5 ml 10X NovaTaq Buffer with MgCl2 •0.2 ml 10 mM dNTP Mix × •1.5 ml 10X NovaTaq Buffer without MgCl2 •5 2ml PCR Grade Water •1.5 ml 10X NovaTaq Optimization Buffer

NOVATAQ PCR MASTER MIX 250 U 71007-3 £119 COMPONENTS: •4 × 1.25 ml 2X NovaTaq PCR Master Mix

•1.5 ml 25 mM MgCl2 •3 × 2 ml PCR Grade Water

NOVATAQ HOT START DNA POLYMERASE 250 U 71091-3 £85 5 x 250 U 71091-4 £370 COMPONENTS: • 250 U or 5 x 250 U NovaTaq Hot Start DNA Polymerase (5 U/µl) •1 or 5 × 1.5 ml 10X NovaTaq Hot Start Buffer × •1 or 5 1.5 ml 25 mM MgCl2

TAQ ANTIBODY * 100 µl 71088-3 £128 COMPONENTS: • 100 µl Taq Antibody (concentration 1µg/µl) •1 ml 10X PCR Buffer

Purchase of NovaTaq DNA Polymerase enzymes and kits, KOD DNA Polymerase enzymes and kits, and the One Step RT-PCR Kit are accompanied by a limited license to use these products in the Polymerase Chain Reaction (PCR) process for research use 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.

KOD DNA Polymerases, the One Step RT-PCR Kit and Taq Antibody are manufactured by Toyobo and distributed by Novagen.

KOD XL DNA Polymerase is licensed under US Patent Number 5,436,149 owned by Takara Shuzo Co., Ltd. 25

* These products are not available through Novagen in Japan Prices subject to change O RDERING I NFORMATION

PRODUCT SIZE CAT NUMBER PRICE

SPINPREP™ PCR CLEAN-UP KIT 100 rxn 70976-3 €106 INTRO.SPINPREP PCR CLEAN-UP KIT 20 rxn 70975-3 €26

SPINPREP GEL DNA KIT 100 rxn 70852-3 €106 INTRO.SPINPREP GEL DNA KIT 20 rxn 70958-3 €26

SPINPREP PLASMID KIT 100 rxn 70851-3 €106 INTRO.SPINPREP PLASMID KIT 20 rxn 70957-3 €26

SPINPREP MASTER KIT 40 rxn 71073-3 €58

PELLET PAINT® CO-PRECIPITANT 125 rxn 69049-3 €60 PELLET PAINT NF CO-PRECIPITANT 125 rxn 70748-3 €60 COMPONENTS: • 250 µl Pellet Paint or Pellet Paint NF Co-Precipitant •1 ml 3 M Sodium Acetate, pH 5.2

PSTBLUE-1 PERFECTLY BLUNT® CLONING KITS 10 rxn 70184-3 €105 20 rxn 70191-3 €196 40 rxn 70191-4 €327

PT7BLUE-3 PERFECTLY BLUNT CLONING KITS 10 rxn 70075-3 €105 20 rxn 70182-3 €196 40 rxn 70182-4 €327

PT7BLUE-2 PERFECTLY BLUNT CLONING KITS 10 rxn 70185-3 €105 20 rxn 70190-3 €196 40 rxn 70190-4 €327

PT7BLUE PERFECTLY BLUNT CLONING KITS 10 rxn 70183-3 €105 20 rxn 70189-3 €196 40 rxn 70189-4 €327

PETBLUE™-1 PERFECTLY BLUNT CLONING KITS 10 rxn 70633-3 €116 20 rxn 70634-3 €219 40 rxn 70634-4 €360

PETBLUE-2 PERFECTLY BLUNT CLONING KITS 10 rxn 70635-3 €116 20 rxn 70636-3 €219 40 rxn 70636-4 €360

PSTBLUE-1 ACCEPTOR™ VECTOR KITS 10 rxn 70594-3 €105 20 rxn 70595-3 €196 40 rxn 70595-4 €327

PETBLUE-1 ACCEPTOR VECTOR KITS 10 rxn 70597-3 €116 20 rxn 70598-3 €219 40 rxn 70598-4 €360

26 PRODUCT SIZE CAT NUMBER PRICE

NOVABLUE SINGLES™COMPETENT CELLS 11 rxn 70181-3 €86 22 rxn 70181-4 €165 COMPONENTS: • 11 × 50 µl or 22 × 50 µl Competent Cells • 10 µl Test Plasmid •2 × 2 ml or 4 × 2 ml SOC Medium

HT96™ NOVABLUE COMPETENT CELLS 1 plate 71011-3 €494 4 plates 71011-4 €1804 20 plates 71011-5 €6872 COMPONENTS: • 96 × 20 µl or 4 × (96 × 20 µl) Competent Cells or 20 × (96 × 20 µl) • 10 µl or 2 × 10 µl or 10 × 10 µl Test Plasmid • 14 ml or 4 × 14 ml or 20 × 14 ml SOC Medium • pkg/l2 or 4 × pkg/l2 or 20 × pkg/l2 8 Cap Strip •1 or 4 or 20 Reagent Reservoir

HT96 ISOTHERMAL BLOCK 1 ea 71031-3 €84

PERFECT DNA™ 50 BP LADDER 100 lanes 70538-3 €99 PERFECT DNA 100 BP LADDER 100 lanes 70539-3 €99 PERFECT DNA 1 KB LADDER 100 lanes 70537-3 €99

PERFECT DNA MARKERS,0.5–12 KBP 100 lanes 69002-3 €99 PERFECT DNA MARKERS,0.1–12 KBP 100 lanes 70087-3 €99 PERFECT DNA MARKERS,0.05–10 KBP 100 lanes 70540-3 €99

PCR MARKERS, 50–2000 BP 50 lanes 69278-3 €75

All Markers include 6X Loading Buffer

27 For a complete listing of kit components, please refer to Novagen’s website: www.novagen.com Prices subject to change United States Novagen, Inc. VWR International & Canada 601 Science Drive Tel. 800 932 5000 Madison,WI 53711 Fax 800 668 6348 Web www.vwr.com Fax 608 238 1388 Te l. 608 238 6110 Te l. 800 526 7319 e-mail [email protected] Web www.novagen.com

United Kingdom CN Biosciences (UK) Ltd. VWR International Ltd Boulevard Industrial Park Hunter Boulevard Padge Road, Beeston Magna Park Nottingham NG9 2JR Lutterworth LE17 4XN

Te l. 0115 9430840 Freephone 0800 223344 Fax 0115 9430951 Fax 01455 558586 e-mail [email protected] Sales e-mail [email protected] Web www.cnuk.co.uk Web www.vwr.com

Germany Calbiochem-Novabiochem GmbH VWR International GmbH Ober der Röth 4, 65824 Schwalbach/Ts. D-64271 Darmstadt Ordering: Freecall 0800 69 31 000 Tel. 0180 570 2000 Freefax 0800 62 36 100 Fax 0180 570 2222 e-mail [email protected] e-mail [email protected] Web www.cnbi.de Technical Service: Freecall 0800 6100 34 96 e-mail [email protected]

NOVAGEN PCR TOOLS www.novagen.com technical support - 800.207.0144

Novagen is a brand of An Affiliate of Merck KGaA, Darmstadt, Germany