TaKaRa Ex Taq 1PCR PRODUCTS. High Performance PCR. Kit Components RR001A. Applications RR01AM. Description. Concentration. Form. Storage.

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1 1PCR PRODUCTS TaKaRa Ex Taq TaKaRa Ex Taq Cat.# RR001A 250 U TaKaRa Ex Taq Cat.# RR001B 1000 U (4 250 U TaKaRa Ex Taq Cat.# RR001C 3,000 U ( U TaKaRa Ex Taq (Mg 2+ free Buffer Cat.# RR01AM 250 U TaKaRa Ex Taq (Mg 2+ free Buffer Cat.# RR01BM 1,000 U (4 250 U TaKaRa Ex Taq (Mg 2+ free Buffer Cat.# RR01CM 3,000 U ( U High Yield and High Sensitivity PCR Improved Fidelity PCR Robust PCR Reproducible PCR Results Excellent on Difficult Templates Takara s Ex Taq combines the proven performance of Takara Taq with an efficient 3 g5 exonuclease activity for unsurpassed PCR performance. In routine PCR applications, the Ex Taq Polymerase and Ex Taq Buffer system gives higher yields and lower mutation rates (approximately 4.5X lower, as determined by the Kunkel method than standard Taq DNA Polymerase. The system also allows amplification of longer products than Taq DNA Polymerase, with 20 kb lengths possible from genomic DNA and up to 30 kb possible from l DNA. Concentration 5 units/µl Form 20 mm Tris HCl (ph 8.0, 100 mm KCl, 0.1 mm EDTA, 1 mm DTT, 0.5% Tween 20, 0.5% Nonidet P-40, 50% Glycerol Purity Nicking activity, endonuclease and exonuclease activity are not detected after the incubation of 0.6 µg of supercoiled pbr322 DNA, 0.6 µg of λ DNA or 0.6 µg of λ-hind III digest with 10 units of this enzyme for 1 hour at 74 C. PCR Performance Test PCR performance by Polymerase Chain Reaction (PCR is confirmed by using λ DNA as the template (amplified fragment: 20 kb. PCR performance is also confirmed via amplification of the β globin gene from a human DNA template. (amplified fragment : 17.5 kb. Most PCR products amplified with TaKaRa Ex Taq have one A added at the 3 -termini, so PCR products can be directly used for cloning into T-vector. Also it is possible to clone the product into blunt-end vectors after blunting and phosphorylation of the end. Approximately 80% of the clones have a 3 A overhang. RR001A TaKaRa Ex Taq 250 U (5 U/µL 10X Ex Taq Buffer (contains 20 mm MgCl 2 dntp Mixture (2.5 mm each dntp 800 µl RR01AM TaKaRa Ex Taq 250 U (5 U/µL 10X Ex Taq Buffer (without MgCl 2 20 mm MgCl 2 dntp Mixture (2.5 mm each dntp 800 µl Amplification of a 6 kb Target from E. coli Genomic DNA with Takara Taq and Takara Ex Taq. PCR was performed using the indicated amounts of template, with either Takara Taq (Lanes T1-T4 or Takara Ex Taq (Lanes E1-E4. Each 50 µl reaction contained 1.25 units of enzyme. Lanes M contain l Hind III DNA Markers. 8

2 EmeraldAmp Max PCR Master Mix EmeraldAmp Max PCR Master Mix Cat.# RR320A 160 reactions (50 µl EmeraldAmp Max PCR Master Mix Cat.# RR320B 800 reactions (50 µl Used for Loading Samples Directly onto a Gel Immediately Following PCR Reaction Features High Yield: yields at least 10X more end product than Taq and can amplify targets up to 10 kb Excellent Performance: optimized buffer for better performance on GC or AT Rich Targets Convenient: just add template, primers and water to 2X Master Mix Eliminate Purification Steps: load PCR reaction directly onto a gel or use for TA cloning or direct sequencing without purification Emerald Green Loading Dye: track migration during electrophoresis Restriction Enzyme Digestion: digest PCR products directly in the PCR buffer Takara s EmeraldAmp Max PCR Master Mix includes an optimized buffer, PCR enzyme, dntp mixture, gel loading dye (green and a density reagent in a 2X premix. Only primer and DNA template need to be added to easily complete the reaction. The mix can be used for routine PCR applications, and PCR reactions performed with this mix can be loaded directly onto a gel for electrophoresis. This product can amplify targets up to ~10kb (genomic DNA and will work well on AT-rich or GC-rich templates. This premix can also yield at least 10X more end product than conventional PCR and has improved performance over standard Taq. The EmeraldAmp Max PCR Master Mix offers a powerful, flexible and convenient alternative for high yield, simple PCR applications. If 5 μl of the reaction mixture is used for electrophoresis with 1% Agarose L03 (Cat.# 5003, the blue dye marker is detected near 3~5 kb and the yellow is below 50 bp. Those dyes have absorptions at around 260nm and 420nm, respectively, so it is recommended to remove the dye by cutting out the gel or extracting DNA by NucleoSpin Extract II (Macherey-Nagel Cat.# /.250, for more applications. Most PCR products amplified with EmeraldAmp Max PCR Master Mix have one A added at the 3 -termini, so PCR products can be directly used for cloning into T-vector. Also it is possible to clone the product into blunt-end vectors after blunting and phosphorylation of the end. Approximately 80% of the clones have a 3 A overhang. RR320A (for 160 reactions, 50 µl each EmeraldAmp Max PCR Master Mix (2X Premix dh 2 O EmeraldAmp Max PCR Master Mix Company D Company E M M M M 4 4 Template: Human Genomic DNA Target: 1. BCL2 581 bp (GC 67.7% 2. IGFB bp (GC 72.3% 3. jun 2025 bp (GC 65.2% 4. IGF2R 3890 bp (GC 63.1% PCR PRODUCTS 1 Comparison of EmeraldAmp Max with Competitors for GC Rich Targets. Takara s EmeraldAmp Max PCR Master Mix was tested against Company D s and Company E s dye-added premixes. EmeraldAmp Max PCR Master Mix had greater reaction efficiency using GC rich targets than Company D s or Company E s dye-added premixes, resulting in significantly higher yields. In addition, EmeraldAmp Max PCR Master Mix provided greater specificity than Company D s dye-added premix. Premix Taq (Ex Taq Version Premix Taq (Ex Taq Version Cat.# RR003A 120 reactions (6 500 µl Premix Taq is an optimized mixture composed of enzyme (TaKaRa Ex Taq, reaction buffer and dntp mixture at a 2-fold concentration. Premix Taq (Ex Taq Version is designed to allow quick and easy assembly of the reaction mixture. RR003A Takara Ex Taq Premix* µl *Contains Takara Ex Taq, Ex Taq Buffer, 4 mm MgCl and dntps. 9

3 1PCR PRODUCTS TaKaRa Ex Taq, Hot-Start Version TaKaRa Ex Taq Hot Start Version Cat.# RR006A 250 U TaKaRa Ex Taq Hot Start Version Cat.# RR006B 1000 U (4 250 U Premix Ex Taq Hot Start Version Cat.# RR030A 100 reactions Robust, Specific Amplification with Reduced Background Amplifications Requiring Room-Temperature Reaction Assembly TaKaRa s Ex Taq HS includes a monoclonal antibody to Taq Polymerase which binds to the polymerase until the temperature is elevated. The binding of this antibody prevents nonspecific amplification due to mispriming and/or formation of primer dimers during reaction assembly. The antibody is then denatured in the initial PCR DNA-denaturation step, releasing the polymerase and allowing DNA synthesis to proceed. TaKaRa Ex Taq HS offers the same high performance as the standard TaKaRa Ex Taq including high yield, excellent sensitivity and fidelity 4.5X better than Taq Polymerase, along with the advantages of hot-start: lower background, increased specificity and room temperature reaction assembly. PCR Performance Enzyme performance is confirmed by successful amplification of a 20 kb l DNA template. Amplification of a single copy gene (b-globin by PCR is also confirmed using human genomic DNA as the template (am plified frag ment: 17.5 kb. Inhibition of TaKaRa Ex Taq activity by the antibody is confirmed to be more than 90% after an incubation at 55 C for 10 min. Company A Takara Ex Taq M M M Amplification Efficiency Comparison Between TaKaRa s Ex Taq HS and a High-Grade Hot Start PCR Enzyme from Company A. A 7.5 kb human genomic DNA target was amplified from increasing amounts of template DNA. The results demonstrate the excellent sensitivity and yield of the TaKaRa Ex Taq HS. Lane M: l-hind III digest, Lane 1: 100 pg, Lane 2: 300 pg, Lane 3: 1 ng, Lane 4: 3 ng, Lane 5: 10 ng, Lane 6: 30 ng, Lane 7: 100 ng. PCR products generated with TaKaRa Ex Taq HS contain a mixture of 3 -A overhangs and blunt ends. Cloning efficiency with T-vectors typically exceeds 80%. RR006A TaKaRa Ex Taq HS 250 U (5 U/µL 10X Ex Taq Buffer (contains 20 mm MgCl 2 dntp Mixture (2.5 mm each dntp 800 µl Use 1.25 U per 50 µl reaction. RR030A TaKaRa Ex Taq HS, Premix** µl ** Contains TaKaRa Ex Taq HS, 1.25 units/25 µl; dntp Mixture, 2X conc.; ea 0.4 mm Ex Taq Buffer, 2X conc.; including 4 nm Mg 2+.. Avoid repeated freeze-thaws and vigorous stirring. Once thawed, aliquot into tubes and store at. Related Products Taq Antibody (Cat.# 9002A Amplification of a 1.1 kb Bacillus sp. Genomic DNA Target. Lanes 1 and 2, TaKaRa Ex Taq HS; lanes 3 and 4, Company A; lanes 5 and 6, Company A with 10X Company A Buffer; lanes 7 and 8, Company C Polymerase; lanes 9 and 10, Company I Taq; lanes 11 and 12 Company B DNA Polymerase kb PerfectShot Ex Taq (Loading Dye Mix PerfectShot Ex Taq (Loading Dye Mix Cat.# RR005A 48 reactions (48 25 µl Direct Loading of Sample on to a Gel after PCR Amplification PerfectShot Ex Taq is a convenient 2X PCR solution (supplied in a 1.2 ml PCR tube which contains Takara s high performance Takara Ex Taq, reaction buffer, dntp mixture and loading dye (2-fold concentration. This premix allows direct loading of the amplified reaction mix onto a TAE gel without the addition of a separate gel loading buffer. Use of high yield Ex Taq plus the convenience of direct gel loading results in consistent, reproducible results for any application. RR005A PerfectShot Ex Taq* 1.2 ml * Contains Ex Taq DNA Polymerase (1.25 U/25 µl, dntp (0.4 mm ea., Ex Taq Buffer (2X, and Loading Dye (Orange G/Bromophenol Blue.. Thaw vials just prior to use. Aliquot to avoid repeated freeze-thaws. 10

4 EmeraldAmp Max HS PCR Master Mix EmeraldAmp Max HS PCR Master Mix Cat.# RR330A 160 reactions (50 µl EmeraldAmp Max HS PCR Master Mix Cat.# RR330B 800 reactions (50 µl Used for Loading Samples Directly onto a Gel Immediately following PCR Reaction Features Antibody Mediated Hot Start: minimize non-specific amplification and primer dimers High Yield: yields at least 10X more end product and can amplify targets up to 15 kb Excellent Performance: optimized buffer for better performance on GC-rich or AT-rich targets Convenient: just add template, primers and water to 2X Master Mix Eliminate Purification Steps: load PCR reaction directly onto a gel Emerald Green Loading Dye: track migration during electrophoresis Restriction Enzyme Digestion: digest PCR products directly in the PCR buffer Takara s EmeraldAmp Max HS PCR Master Mix includes a high yield, hot start PCR enzyme, optimized buffer, dntp mixture, gel loading dye (green and a density reagent in a 2X premix. Only primer and DNA template need to be added to easily complete the reaction. The mix can be used for routine PCR applications, and PCR reactions performed with this mix can be loaded directly onto a gel for electrophoresis. This product can amplify targets up to ~15kb (genomic DNA and will work well on high AT or GC templates. This premix can also yield at least 10X more end product than conventional PCR and has an improved performance over standard Taq. The EmeraldAmp Max HS PCR Master Mix offers a powerful, flexible and convenient alternative for high yield, simple PCR applications. If 5 μl of the reaction mixture is used for electrophoresis with 1% Agarose L03 (Cat.# 5003, the blue dye marker is detected near 3~5 kb and the yellow is below 50 bp. Those dyes have absorptions at around 260nm and 420nm, respectively, so it is recommended to remove the dye by cutting out the gel or extracting DNA by NucleoSpin Extract II (Macherey-Nagel Cat.# /.250 for more applications. RR330A (for 160 reactions, 50 µl each EmeraldAmp Max PCR Master Mix dh 2 O (2X Premix 4 4 PCR PRODUCTS 1 EmeraldAmp Max Company F Company F HS PCR Master Mix w/o dye w/ dye Company B Company A M M M M M M Template: Human Genomic DNA Target: 1. BCL2 581bp (GC 67.7% 2. IGFB-1 987bp (GC 72.3% 3. jun 2025bp (GC 65.2% 4. IGF2R 3890bp (GC 63.1% Template Amount: 50 ng Reaction Size: 25 µl Comparison of EmeraldAmp Max HS with Competitors for GC Rich Targets. Takara s EmeraldAmp Max HS PCR Master Mix was tested against Company A s, Company B s and Company F s dye-added premixes, as well as Company F s premix without dye. EmeraldAmp Max HS PCR Master Mix showed the best reaction efficiency (with significantly higher yields compared to the other premixes and was the only premix able to amplify the 3890 bp fragment. Taq Antibody Taq Antibody Cat.# 9002A 250 U Taq Antibody Cat.# 9002B 1000 U (4 250 U Increasing Specificity and Lowering Background in PCR Reactions Room Temperature Reaction Assembly of PCR Reactions The Taq antibody is a monoclonal antibody to Taq Polymerase which binds to the polymerase until the temperature is elevated. The binding of this antibody prevents nonspecific amplification due to mispriming and/or formation of primer dimers during PCR reaction assembly. The antibody is denatured in the initial PCR DNAdenaturation step, releasing the polymerase and allowing DNA synthesis to proceed. The addition of this antibody to Taq Polymerase, TaKaRa Ex Taq or before a PCR reaction lowers background, increases specificity and allows room temperature reaction assembly. Concentration 5 U/µL Antibody Performance Inhibition of Taq polymerase activity by the antibody was confirmed to be more than 90% after incubation at 55 C for 10 min, after the incubation of the mixture of Taq Antibody and Taq Polymerase at 25 C for 10 min. Taq Polymerase activity was confirmed to recover almost 100% activity in a reaction at 55 C for 10 minutes after heat treatment at 94 C for 30 seconds. 11

5 1PCR PRODUCTS (without Mg 2+ Cat.# RR002A 125 U Cat.# RR002M 250 U DNA Polymerase Cat.# RR002B 1,000 U (4 250 U DNA Polymerase Cat.# RR002C 3,000 U ( U Robust Amplification of Long DNA Templates (up to 48 kb possible with Minimal Optimization Longer and More Accurate Amplification of Genomic is a mixture of Taq Polymerase with a proofreading polymerase optimized for amplification of long DNA templates. Using, routine extension to 20 kb and up to 48 kb is possible (depending on template type with less optimization than other long PCR systems. Because of the presence of the proofreading polymerase, the fidelity is significantly better (6.5X than that of Taq Polymerase alone. RR002M 250 U (5 U/µL* 10X LA PCR Buffer II (contains 25 mm MgCl 2 dntp Mixture (2.5 mm each dntp 800 µl RR002A 125 U (5 U/µL* 10X LA PCR Buffer II (without Mg mm MgCl 2 dntp Mixture (2.5 mm each dntp 400 µl * Use 2.5 U per 50 µl reaction. PCR products generated with contain a mixture of 3 -A overhangs PCR Performance Enzyme performance is confirmed by successful amplification of a 35 kb l DNA template and a 17.5 kb human genomic DNA template.. Avoid repeated freeze-thaws of and dntps. Once thawed, aliquot into separate tubes and store at. M M2 M1: phy Marker 1: 0.5 kb 2: 1 kb 3: 2 kb 4: 4 kb 5: 6 kb 6: 8 kb 7: 10 kb 8: 12 kb 9: 15 kb 10: 20 kb 11: 28 kb 12: 35 kb M2: λ-hind III digest Amplification of l DNA Fragments from kb in Size (different primer sets using. was used to amplify the various fragments and generated high product yields, even with very long (28 kb fragments. One-Shot LA PCR Mix One-Shot LA PCR Mix Cat.# RR tubes (25 µl ea. Convenient, Reliable Amplification of Long DNA Fragments Longer and More Accurate Genomic PCR Amplifications Increased Reproducibility and Minimal Optimization for Long PCR Excellent for High-Throughput Studies The One-Shot LA PCR Mix is an optimized mixture composed of, Buffer and dntps at a 2X concentration in 25 µl aliquots. These aliquots are predispensed into 0.2 ml PCR tubes. By addition of an equal volume of solution containing the template and primers to each tube, a complete, ready-to-amplify reaction is obtained. RR004 One-Shot LA PCR Mix* µl *Contains, LA PCR Buffer II (2X conc.; including 5 mm Mg 2+ and dntps. PCR products generated with contain a mixture of 3 -A overhangs 12

6 LA PCR Kit Version 2.1 LA PCR Kit Version 2.1 Cat.# RR013A 50 reactions LA PCR Kit Version 2.1 Cat.# RR013B 100 reactions Amplification of Large DNA Templates (30 kb genomic or 48 kb l DNA Longer and More Accurate Genomic Includes GC buffers at 2X Concentration for Templates of High GC-Content with Strong Secondary Structure The LA PCR Kit, Ver. 2.1 includes all the reagents necessary for amplification of long DNA templates with routine extension to 30 kb (up to 48 kb is possible with some templates. The Version 2.1 kit combines with an optimized buffer system resulting in longer and more accurate PCR products than conventional PCR reagents. High fidelity (6.5-fold better than conventional Taq DNA Polymerase is facilitated by an efficient 3 g5 exonuclease activity. The Version 2.1 kit contains a control template and primers to ensure premium PCR performance. Amplification of Human Genomic DNA with. Purified human genomic DNA (500 ng in a 50 µl reaction was used as template for PCR with. The primer sets used amplified target regions in the b-globin gene cluster and the TPA gene. The sizes of the amplified products were 17.5 kb (lane 1, 21.5 kb (lane 2, and 27 kb (lane 3. Lane M contains High MW Markers (Life Technologies. Includes Everything Needed for Long PCR Optimization M kb kb kb RR013A 125 U (5 U/µL 10X LA PCR Buffer II (contains 25 mm Mg µl 10X LA PCR Buffer II (without Mg µl MgCl 2 (25 mm 500 µl dntp Mixture (2.5 mm each dntp 400 µl Control Template (100 ng/µl HT29 DNA 10 µl Control Primer LA3 (10 pmol/µl* 10 µl Control Primer LA4 (10 pmol/µl* 10 µl λ-hind III MW Markers (100 ng/µl 20 µl 2X GC Buffer I (contains 5 mm MgCl ml 2X GC Buffer II (contains 5 mm MgCl ml Control Primer GC1 (10 pmol/µl** 10 µl Control Primer GC2 (10 pmol/µl** 10 µl * Amplifies a 17.5 kb region of the Control Template. ** Amplifies a 1,255 bp GC-rich region of the Control Template. PCR products generated with contain a mixture of 3 -A overhangs PCR PRODUCTS 1 13

7 1PCR PRODUCTS Hot-Start Version Hot Start Version Cat.# RR042A 125 U Hot Start Version Cat.# RR042B 500 U (4 125 U Long Amplifications Synthesizing Products up to 30 kb (genomic DNA and 48 kb (l DNA High Fidelity Amplifications (6.5X higher than Taq DNA Polymerase Enzyme-Buffer System Allows Robust Amplification and High Yield with Less Need for Optimization Reduced Background and Increased Reaction Specificity Compatible with Multiplex PCR Hot-Start consists of plus a monoclonal antibody to Taq Polymerase. It retains all of the high performance features of TaKaRa LA Taq and provides increased reaction specificity and reduced background. The key features of are: synthesis of products up to 30 kb (genomic DNA and 48 kb (l DNA, 6.5X higher fidelity than Taq DNA Polymerase, and less optimization and greater product yield than other long polymerases due to the robust enzyme-buffer system. Room temperature reaction assembly is possible with this formulation. RR042A HS 125 U (5 U/µL* 10X LA PCR Buffer II (Mg mm dntp Mixture (2.5 mm each dntp 400 µl * Use 2.5 U per 50 µl reaction. PCR products generated with contain a mixture of 3 -A overhangs PCR Performance Performance is confirmed by successful amplification of a 35 kb l DNA template and a 17.5 kb human genomic DNA. Inhibition of activity by the anti-taq antibody (hot-start formulation is confirmed to be more than 90% after an incubation of 55 C for 10 min.. Avoid repeated freeze-thaws of and dntps. Once thawed, aliquot into separate tubes and store at. LA PCR Genome DNA Set LA PCR Genome DNA Set Cat.# reactions Human Genomic and E. coli Genomic DNA Control Templates and Primers for Use with the LA PCR Kit This set includes highly purified, high molecular weight human genomic DNA and E. coli genomic DNA, along with appropriate primers. These templates and primers are intended for use as controls for the LA PCR Kit and are useful for optimizing PCR conditions for a variety of templates Human genomic DNA 10 µg E. coli genomic DNA 2 µg Human-1 Primer* 200 pmol Human-2 Primer* 200 pmol E. coli-1 Primer** 200 pmol E. coli-2 Primer** 200 pmol * Amplimer size: 17.5 kb ** Amplimer size: 20 kb DNA: 4 C (Can be stored at. Avoid freeze-thaw cycles. Primers: 14

8 with GC Buffers with GC Buffers Cat.# RR02AG 125 U Amplification of Large DNA Templates (up to 48 kb possible Longer and More Accurate Genomic PCR Amplification Amplification of GC-rich Templates is a mixture of Taq Polymerase with a proofreading polymerase optimized for amplification of long DNA templates. Using, routine extension to 20 kb is possible (up to 48 kb on some templates, with less optimization than other long PCR systems. Because of the presence of the proofreading polymerase, fidelity is significantly better (6.5X than Taq Polymerase alone. The GC-optimized Buffers I and II are specifically designed for DNA templates with high GC content or a significant amount of secondary structure. GC Buffer I is recommended for amplification of fragments $5kb. GC Buffer II is recommended for 2 3 kb fragments with up to 73% GC content. RR02AG 125 U (5 U/µL* 2X GC Buffer I (contains 5 mm MgCl ml 2X GC Buffer II (contains 5 mm MgCl ml dntp Mixture (2.5 mm each dntp 400 µl * Use 2.5 U per 50 µl reaction. PCR products generated with contain a mixture of 3 -A overhangs PCR Performance Performance is confirmed by successful amplification of a 35 kb l DNA template, and a 17.5 kb human genomic DNA with GC Buffer I. Amplification of the c jun proto oncogene (1,255 bp, 65% GC content by PCR is also confirmed using human genomic DNA as the template with GC Buffer I and GC Buffer II.. Avoid repeated freeze-thaws of and dntps. Once thawed, aliquot into separate tubes and store at. PCR PRODUCTS 1 M M2-2.1 kb Comparison of Amplification Efficiency between with GC Buffer and using a GC-rich Target Fragment. A 2.1 kb mouse rrna gene with 63.4% GC was amplified from 1 ng of mouse genomic DNA. The results show with GC Buffer gave optimal results in amplification of the 2.1 kb fragment with both excellent yields and high specificity. M1.: l -Hind III digest 1 & 2: LA Taq / LA PCR Buffer II 3 & 4: LA Taq with GC Buffer/ GC Buffer I 5 & 6: LA Taq with GC Buffer/ GC Buffer II M2: phy Marker M kb -262 kb Amplification of a Huntington s Disease Gene (high GC content. Purified human genomic DNA (100 ng in a 50 µl reaction was used as a template for PCR with TaKaRa LA Taq and either LA PCR Buffer II (lane 1, GC Buffer I (lane 2, or GC Buffer II (lane 3, or with a competing DNA Polymerase and high GC Kit (lane 4. The primers amplified regions of the HD gene IT15 CAG repeat. The sizes of the amplified products were 262 bp (GC content 73%, and 358 bp (GC content 71.5%. Lane M contains a 100 bp ladder. 15

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