High Yield/ Routine Applications Problematic templates, High GC/AT High Fidelity Heat-Activated Long Products High Specificity DHPLC Compatible

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1 Yield/ Routine Applications Problematic templates, GC/AT Fidelity Heat-Activated Long Products Specificity DHPLC Compatible Polymerases Optimised dntp/ Polymerase Mixes

2 Introduction are essential tools in today s molecular biology. The number of commercially available polymerases has grown rapidly in the last few years, making it sometimes difficult to choose the correct enzyme required for each experiment. Bioline has been serving the scientific community for 15 years and has substantial expertise in this area of molecular biology. Each polymerase has different characteristics and choosing the enzyme that suits your individual experiment is crucial. Bioline polymerases are available separately but are also supplied as convenient ready-to-go 2x reaction mixes, containing the polymerase of choice, dntps*, MgCl 2 and further additives to achieve optimal reaction conditions. Bioline Mixes (page 10) only require the user to add water, template and primers. Using these optimised mixes, the amount of time needed to set up reactions and the risk of contamination are dramatically reduced. The number of pipetting steps is also minimised, which ensures greater experiment reproducibility. This brochure aims to simplify your polymerase/ polymerase mix selection process. However, should you need further guidance in choosing the correct enzyme for your experiment, please contact tech@bioline.com *Note: Bioline polymerase mixes contain our inhouse-manufactured ultra-pure dntps. Bioline s >99% pure nucleotides help you to succeed in your experiments. For further information on dntps, please us requesting our dntp brochure. Yield/ Routine Applications BIOTAQ p 02 BIOTAQ Red p 02 Heat Activated IMMOLASE p03 AccuSure p06 DH-HotPol p 08 Specificity Diamond p 07 IMMOLASE p 03 AccuSure p 06 Problematic Templates, GC/AT BIOXACT Short p 04 BIOXACT Long p 04 Diamond p 07 The EasyGuide to DNA Polymerases DHPLC Compatible DH-HiPol p 08 DH-HotPol p 08 Fidelity ACCUZYME p 05 AccuSure p 06 BIOXACT Short p 04 BIOXACT Long p 04 DH-HiPol/ HotPol p 08 Long Products BIOXACT Long p 04 dntp/ Polymerase Mixes (see page 10) 01

3 Yield/ Routine Apps. BIOTAQ/ BIOTAQ Red Robust performance in a wide range of applications. Leave an A overhang. Separate MgCl 2 solution supplied. ly suited to high-throughput applications. Direct loading onto agarose gels (BIOTAQ Red only). Easy visual recognition (BIOTAQ Red only). BIOTAQ/ BIOTAQ Red are highly purified thermostable DNA polymerases offering a very high yield over a wide range of templates and they are the ideal choice for most assays. Both BIOTAQ & BIOTAQ Red possess 5-3 exonuclease activity and leave an A overhang such that the primer extension product is suitable for effective integration into TA cloning vectors. BIOTAQ Red is an ideal choice for highthroughput applications, since it contains an inert red dye to facilitate easy recognition. When the reaction is complete, a sample of the reaction mix can be loaded directly onto agarose gels without the need for loading buffer. This enzyme is supplied at a concentration of 1 u/µl. BIOTAQ Core Kit Ideal for labs setting up new protocols. The BIOTAQ Core Kit contains all the individual reagents necessary to perform polymerase assays on a wide range of DNA templates. The Core Kit is based on our widely used BIOTAQ, which offers dependable high-performance in routine or high-throughput assays. Additionally, the kit includes dntp mix, buffer, MgCl 2 and the special PolyMate Additive for difficult reactions. BioMix/ BioMix Red Both BIOTAQ and BIOTAQ Red are also available as convenient ready-to-go 2x reaction mixes, Biomix and Biomix Red respectively. The user only needs to add water, template and primers. BIOTAQ DNA Polymerase 5 u/µl 100 Units BIO BIOTAQ DNA Polymerase 5 u/µl 500 Units BIO BIOTAQ DNA Polymerase 5 u/µl 2500 Units BIO BIOTAQ Red DNA Polymerase 1 u/µl 100 Units BIO BIOTAQ Red DNA Polymerase 1 u/µl 500 Units BIO BIOTAQ Red DNA Polymerase 1 u/µl 2500 Units BIO BIOTAQ Taq Core Kit _ 500 Units BIO BioMix 2x 100 Reactions BIO BioMix 2x 500 Reactions BIO BioMix Red 2x 100 Reactions BIO BioMix Red 2x 500 Reactions BIO

4 Heat Activated Specificity SPECIAL OFFER Buy IMMOLASE for the price of Taq! IMMOLASE DNA Polymerase Heat-activated, inactive at room temperature. Ultra high specificity (eliminates non-specific reaction products). Leaves A overhang. Activity Time (minutes) 97 C 95 C 93 C Activation is most effective at 95 C for 7-10 minutes. Activity PACK SIZE CONC. CAT NUM. PRICE 50 Reactions 10u/µl BIO Reactions 0 10u/µl BIO Temperature ( C) IMMOLASE DNA Polymerase Taq DNA Polymerase Wrongly annealed primers could be extended in this temperature range The illustration compares Taq during its active state with a potential to extend wrongly annealed primers, whereas IMMOLASE is still inactive. IMMOLASE is a heat-activated thermostable DNA polymerase isolated from a novel organism. IMMOLASE provides improved specificity when compared to standard polymerases and can eliminate the presence of non-specifics, such as primer-dimers and mis-primed products. Activation profile for IMMOLASE DNA Polymerase IMMOLASE is inactive at room temperature and must therefore be activated by heat treatment before primer extension. ImmoMix/ ImmoMix Red ImmoMix/ Immomix Red are complete ready-to-go heat-activated 2x reaction mixes which only require the user to add water, template and primers and then pre-heat to 95 C. Both mixes contain IMMOLASE DNA Polymerase, dntps, MgCl 2 and further additives to achieve optimal reaction conditions. In addition, ImmoMix Red contains an additional non-toxic, non-hazardous red dye, allowing users to load samples directly onto a gel, without the need to add loading buffer. Adequate mixing is also ensured when reactions are set up. PRODUCT PACK SIZE CAT NO. IMMOLASE (4 u/µl) 250 Units BIO IMMOLASE (4 u/µl) 500 Units BIO IMMOLASE (4 u/µl) 5000 Units BIO PRODUCT PACK SIZE CAT NO. 2x ImmoMix 100 Reactions BIO x ImmoMix 500 Reactions BIO x ImmoMix Red 100 Reactions BIO x ImmoMix Red 500 Reactions BIO

5 Problem. templates GC/AT Fidelity Long Products (BIOXACT Long) BIOXACT BIOXACT Short Suited to problematic templates such as GC-rich or dirty DNA. Possess 5-3 polymerase activity and 3-5 proof-reading activity, for enhanced fidelity. BIOXACT Short and BIOXACT Long are the polymerases of choice for difficult applications that otherwise fail. BIOXACT DNA polymerases contain a proprietary mix of components, which can be used on almost any template. In addition to spanning difficult regions successfully, BIOX- ACT polymerases offer >17-fold fidelity as compared with Taq. To achieve an optimal ph stability in the reaction, an additional buffer, OptiBuffer, is supplied. For enhanced specificity, BIOXACT polymerases may be used with 2x PolyMate Additive (not supplied). This polymerase has been optimised to maximise its performance with difficult templates up to 3 kb (genomic) or 5 kb (lambda DNA). BIOXACT Long This polymerase has been optimised to maximise its performance with long templates. BIOXACT Long is suitable for genomic fragments of 20Kb or 30Kb lambda fragments. BIOXACT Long is our new version of BIOXACT DNA Polymerase. BIOXACT Short & Long Mixes Complete ready-to-go 2x reaction mixes, which only require the user to add water, template and primers. The mixes contain BIOXACT (Short or Long) DNA Polymerase, dntps, MgCl 2 and further additives to achieve optimal reaction conditions. BIOXACT Short 4 u/µl 250 Units BIO BIOXACT Short 4 u/µl 500 Units BIO BIOXACT Long 4 u/µl 250 Units BIO BIOXACT Long 4 u/µl 500 Units BIO BIOXACT Short Mix 2x 100 Reactions BIO BIOXACT Short Mix 2x 500 Reactions BIO BIOXACT Long Mix 2x 100 Reactions BIO BIOXACT Long Mix 2x 500 Reactions BIO

6 Fidelity ACCUZYME DNA Polymerase ACCUZYME Mix fidelity. Processes fragments up to 5 kb. Produces blunt ends. ACCUZYME is a thermostable enzyme possessing 5-3 DNA polymerase and 3-5 proof-reading exonuclease activities, offering extremely high-fidelity (up to 47- fold higher fidelity than Taq). Accuzyme produces blunt-ended amplicons of up to 5 kb in length. ACCUZYME is supplied with 10x buffer containing MgSO 4, which provides optimal final reaction conditions (2 mm Mg 2 +) for most experiments. In order to allow optimisation of reaction conditions, additional MgCl 2 is also provided. Convenient pre-mixed, pre-optimised solution. Same high fidelity as ACCUZYME. Suitable for high-throughput applications. ACCUZYME is also available as a convenient 2x reaction mix to maximise experiment reproducibility. ACCUZYME Mix contains ACCUZYME DNA Polymerase, ultra-pure dntps manufactured by Bioline and MgCl 2. The Mix is optimised and ready-to-go: the user adds only water, template and primers. ACCUZYME is supplied with an additional 50 mm MgCl 2 solution for optional optimisation of reaction conditions. ACGTAGTCAGT DON'T TAKE A GAMBLE AVOID MISMATCHES GGTAACGTATG I I I I I I I I I I I I I I I I I I I I I I TGCATCAGTCA CCATTGCATAC ACCUZYME 2.5 u/µl 250 Units BIO ACCUZYME 2.5 u/µl 500 Units BIO ACCUZYME Mix 2x 100 Reactions BIO ACCUZYME Mix 2x 500 Reactions BIO

Fidelity Heat Activated Specificity AccuSure DNA Polymerase AccuSure Mix Heat-activated. fidelity. Processes fragments up to 5 kb. Produces blunt ends.

7 Fidelity Heat Activated Specificity AccuSure DNA Polymerase AccuSure Mix Heat-activated. fidelity. Processes fragments up to 5 kb. Produces blunt ends. AccuSure DNA polymerase possesses the same high fidelity as ACCUZYME (page 5) but has the additional advantage of being heat-activated, offering enhanced specificity. AccuSure is ideally suited for difficult templates, such as DNA secondary structures and microsatellites. AccuSure produces blunt-ended amplicons of up to 5 kb in length. AccuSure is also supplied with 10x buffer containing MgSO 4 but, in order to allow optimisation of reaction conditions, additional MgCl 2 is also provided. Convenient pre-mixed, pre-optimised solution. Same high-fidelity and specificity as ACCUZYME. Suitable for high-throughput applications. AccuSure is also available as a convenient 2x Reaction Mix to maximise experiment reproducibility. AccuSure Mix contains AccuSure DNA Polymerase, ultrapure dntps manufactured by Bioline and MgCl 2. The Mix is optimised and ready-togo: the user adds only water, template and primers. AccuSure is supplied with an additional 50 mm MgCl 2 solution for optional optimisation of reaction conditions. ACGTAGTCAGT TGCATCAGTCA DON'T TAKE A GAMBLE AVOID MISMATCHES GGTAACGTATG I I I I I I I I I I I I I I I I I I I I I I CCATTGCATAC AccuSure 2.5 u/µl 250 Units BIO AccuSure 2.5 u/µl 500 Units BIO AccuSure Mix 2x 100 Reactions BIO AccuSure Mix 2x 500 Reactions BIO

recognition for mismatches in allele-specific applications. Suitable for high-throughput and MALDI-TOF spectrometry use.

8 Specificity Problem. templates GC/AT Diamond DNA Polymerase Extremely high specificity (mis-matched primers not extended). Ideal for regions such as high GC, inverted repeats, microsatellites and complex secondary structures. Suited to multiplex, SNP detection, genotyping applications. recognition for mismatches in allele-specific applications. Suitable for high-throughput and MALDI-TOF spectrometry use. Diamond DNA Polymerase is designed for difficult templates such as high GC% regions and microsatellites. When templates are challenging, the enzyme maintains excellent specificity and minimal background, even in conditions such as high concentration of Mg ++, primers and dntps. On genomic templates, Diamond can be used with MgCl 2 concentrations as high as 10mM. Diamond also extends through inverted tandem repeats and regions with high amounts of secondary structure. Diamond possesses improved nucleotide selection and a lower rate of mis-match extension, such that only perfectly aligned primers will be extended (see below). Taq Oligonucleotide ends with mismatch DNA strand Diamond Oligonucleotide ends with mismatch DNA strand Taq Diamond Diamond Mix Taq continues along the DNA strand regardless of mismatch Point of mismatch Diamond recognises mismatch and halts immediately Point of mismatch Diamond Mix is a complete ready-to-go 2x reaction mix for users seeking extremely high specificity in a range of difficult templates. Diamond Mix is highly suited to SNP detection. Diamond 5 u/µl 250 Units BIO Diamond 5 u/µl 500 Units BIO Diamond Mix 2x 100 Reactions BIO Diamond Mix 2x 500 Reactions BIO

9 DHPLC Compatible Fidelity Heat Activated (DH- HotPol) DHPLC Polymerases Compatible with DHPLC columns (detergent-free). Up to 40-fold higher fidelity than Taq. Suitable for mutation detection and genotyping. Products are blunt-ended. There is currently an increasing demand for reagents dedicated to the analysis of nucleic acids by means of Denaturing -Performance Liquid Chromatography (DHPLC), an emerging alternative to gelbased separation. The high-throughput and sensitive nature of this column-based technology demands detergent-free polymerases that are highly purified and give consistently reproducible results. For applications such as detecting mutation, a proof-reading polymerase is also essential. Bioline manufactures 2 DHPLCcompatible DNA polymerases: DH-HiPol and DH-HotPol. In contrast to detergentfree polymerases from other manufacturers, both are single proof-reading enzymes which do not contain or require the addition of non-proofreading polymerase to enhance activity. DH-HiPol DNA Polymerase DH-HiPol is a high-performance proofreading thermostable DNA Polymerase ideal for mutation detection applications. DH-HiPol posseses a 3'-5' proof-reading exonuclease domain, making it up to 40 times more accurate than Taq. DH-HotPol DNA Polymerase Heat-activated enzyme. Ideal for high-throughput applications. DH-HotPol DNA Polymerase is the heatactivated version of DH-HiPol, which possesses 5'-3' DNA polymerase, as well as 3'-5' proof-reading activities to prevent misincorporations. DH-HotPol lacks polymerase activity at reaction set-up temperatures, which dramatically reduces the possibility of misprimed products being accumulated. Both enzymes have also been optimised for use in detergent-free systems, so that suitable conditions are provided for loading samples directly onto DHPLC columns. DH-HiPol 2.5 u/µl 500 Units BIO DH-HotPol 2.5 u/µl 500 Units BIO

10 Other Klenow Fragment No exonuclease activity. Synthesis of the second strand of cdna. Radiolabelling by filling in 5 single-stranded extensions on double-stranded DNA. Generation of blunt-ended DNA fragments. Dideoxy sequencing. Klenow fragment is a proteolytic subfragment of E.coli DNA polymerase I. The enzyme is purified from E.coli PVG-AI strain and is of sequencing grade. T4 DNA Polymerase Possessing 5-3 DNA polymerase and 3-5 proof-reading exonuclease activities. Lacking in 5-3 exonuclease activity. In vitro mutagenesis. Probe-labelling. Removal of 3 overhang to form blunt ends. T4 DNA Polymerase catalyses the synthesis of DNA in the 5-3 direction in the presence of template and primers. This enzyme has 3-5 proof-reading exonuclease activity. Tth Plus DNA Polymerase Reverse transcription and extension in a single coupled reaction. Ideal for difficult templates. Processes both RNA and DNA templates. No RNase activity. Tth Plus is a dual-function polymerase which provides reverse transcription of mrna and subsequent DNA-dependent primer extension in a single coupled reaction at 72 C, so eliminating the need for mesophilic reverse transcriptases such as AMV or MMLV. The thermostable nature of this enzyme is particularly useful when difficulties arise with the RNA secondary structure. This feature provides a clear advantage over the AMV and MMLV Reverse Transcriptases. Klenow Fragment 5 u/µl 500 Units BIO T4 DNA Polymerase 10 u/µl 250 Units BIO T4 DNA Polymerase 10 u/µl 500 Units BIO Tth Plus DNA Polymerase 4 u/µl 250 Units BIO Tth Plus DNA Polymerase 4 u/µl 500 Units BIO

11 Yield/ Routine Apps. Heat Activated Fidelity Problem. templates Specificity GC/AT Long Products Optimised Polymerase/ dntp Mixes Convenient, optimised 2x mixes. Time saving. ly reproducible results. Risk of contamination reduced. Most Bioline DNA polymerases can be supplied as convenient polymerase/ dntp/mg mixes. These are complete ready-to-go 2x reaction mixes, which only require the user to add water, template and primers. Using these optimised mixes, the amount of time needed to set up reactions and the risk of contamination are dramatically reduced. The number of pipetting steps is also minimised, which ensures greater experiment reproducibility. PRODUCT PACK SIZE CAT NO. PAGE 2x BioMix 100 Reactions BIO x BioMix 500 Reactions BIO x BioMix Red 100 Reactions BIO x BioMix Red 500 Reactions BIO x ImmoMix 100 Reactions BIO x ImmoMix 500 Reactions BIO x ImmoMix Red 100 Reactions BIO x ImmoMix Red 500 Reactions BIO x BIOXACT Short Mix 100 Reactions BIO x BIOXACT Short Mix 500 Reactions BIO x BIOXACT Long Mix 100 Reactions BIO x BIOXACT Long Mix 500 Reactions BIO x ACCUZYME Mix 100 Reactions BIO x ACCUZYME Mix 500 Reactions BIO x AccuSure Mix 100 Reactions BIO x AccuSure Mix 500 Reactions BIO x Diamond Mix 100 Reactions BIO x Diamond Mix 500 Reactions BIO

P HENIX. PHENIX PCR Enzyme Guide Tools For Life Science Discovery RESEARCH PRODUCTS

P HENIX. PHENIX PCR Enzyme Guide Tools For Life Science Discovery RESEARCH PRODUCTS PHENIX PCR Enzyme Guide PHENIX offers a broad line of premium quality PCR Enzymes. This PCR Enzyme Guide will help simplify your polymerase selection process. Each DNA Polymerase has different characteristics