High-Fidelity PCR

Product description This kit is a ready-to-use 2×pre-mixed solution containing High-Fidelity DNA Polymerase, dNTPs, and an optimized buffer system. The mix includes two monoclonal antibodies that inhibit polymerase activity and 3’→5’exonuclease activity at room temperature, enabling highly specific Hot Start PCR. The addition of elongation factors allows for long-fragment amplification, with target fragments up to 13 kb. This enzyme exhibits 5’→3’ DNA polymerase activity and 3’→5’ exonuclease activity, with a fidelity 83 times that of Taq DNA polymerase and 9 times that of ordinary Pfu DNA polymerase. It is suitable for amplifying complex templates, producing blunt-end PCR products. The kit offers advantages such as simplicity, high sensitivity, strong specificity, and excellent stability. The reaction system only requires the addition of primers and template, and a two-step PCR protocol can be used to simplify the procedure and save time. The product includes electrophoresis tracking dye, allowing direct loading of PCR products for gel analysis. Additionally, it contains a special protective agent that ensures stable activity even after repeated freeze-thaw cycles.  Components Components N132014S N132014M N132014L 2× High-Fidelity PCR Master Mix (With Dye) 1 mL 5×1 mL 100×1 mL Shipping and Storage Dry ice shipping. -15℃ ~ -25℃ storage, valid for one year. Notes 1. This product is for research use only. 2. Please operate with lab coats and disposable gloves, for your safety. Instructions Recommended PCR reaction systems. Table 1 PCR reaction system Components Volume(μL） Final concentration 2× High-Fidelity PCR Master Mix (With Dye)* 25 1× Template*** x - Forward Primer（10 μmol/L）** 2 0.4 μmol/L Reverse Primer（10 μmol/L）** 2 0.4 μmol/L ddH2O Up to 50 - 【Note】: 1) *The 1× final concentration of the mix contains 1 U/50 μL of polymerase, 2 mmol/L Mg2+, and 200 μmol/L dNTPs.   2) **The recommended final concentration of primers in the PCR reaction is 0.2-1 μmol/L, with 0.4 μmol/L preferred. 3) ***Recommended template amounts for a 25 μL reaction system: Table 2 Recommended template amounts Template Type Fragment Size (1 kb-10 kb) Genomic DNA 50 ng-200 ng Plasmid or Viral DNA 10 pg-20 ng cDNA 1-2.5  μL (not exceeding 1/10 of the total PCR volume) Reaction program. 1）Two-Step Protocol (Preferred):  Cycle step Temp. Time Cycles Initial denaturation 98℃ 3 min 1 Denaturation 98℃ 10 sec   30-35 Extension 68℃ 30 sec/kb Final extension 72℃ 5 min 1 2）Three-Step Protocol (Conventional): Cycle step Temp. Time Cycles Initial denaturation* 98℃ 3 min 1 Denaturation 98℃ 10 sec   30-35 Annealing** 60℃ 20 sec Extension*** 72℃ 30 sec/kb Final extension 72℃ 5 min 1 【Note】: a. * Initial Denaturation: Recommended at 98°C for 3 min (5-10 min for high-GC templates). b. **Annealing: Recommended at 60°C for 20 sec (adjustable between 10-30 sec). Prolonged annealing may cause smearing on gels.  c. ***Extension: Recommended at 72°C for 30 sec/kb (up to 60 sec/kb for complex templates). 3）Gradient Annealing Protocol (Recommended for Difficult Targets): Cycle step Temp. Time Cycles Initial denaturation 98℃ 3 min 1 Denaturation 98℃ 10 sec 15 (decrease by 1° C per cycle) Gradient Annealing 70-55°C 20 sec Extension 72℃ 30 sec/kb Denaturation 98°C 10 sec   20 Annealing 55°C 20 sec Extension 72°C 30 sec/kb Final extension 72℃ 5 min 1 4）Characteristics of Different PCR Protocols: Protocol Type Speed Specificity PCR Yield Detection Rate Two-Step Fastest High Medium High Three-Step Medium Medium Highest Medium Gradient Annealing Slow High Medium High  

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Product description This kit is based on the Pyroccus Furiosis DNA Polymerase, genetically engineered. The enzyme has a 5'→3' DNA polymerase activity and a 3'→5' exonuclease activity, and its fidelity is 83 times that of Taq DNA polymerase and 9 times that of ordinary Pfu DNA polymerase. Two monoclonal antibodies at room temperature that inhibit polymerase activity and 3'→5' exonuclease activity were added to the enzyme solution, which can easily perform highly specific Hot Start PCR, greatly improving the detection rate of amplification and the specificity of the product. The addition of an extension factor to the enzyme solution gives the enzyme the ability to amplify long fragments, and the length of the amplification of the fragment of interest can be up to 13 kb. This product is equipped with an optima buffer that makes the enzyme suitable for amplification of complex templates. It generates blunt ends in the amplification products. Components Components No. Name N132015S (100 U) N132015M (500 U) N132015L (1000 U) N132015-A High-Fidelity DNA Polymerase (1 U/μL) 100 μL 500 μL 500 μL×2 N132015-B 2×PCR buffer（with Mg2+，dNTPs） 3×1 mL 15×1 mL 30×1 mL N132015-C 6×DNA Loading Buffer 1 mL 6×1 mL 12×1 mL Applications Gene cloning; amplification of complex templates DNA; high-throughput library building. Shipping and Storage Dry ice shipping. -15℃ ~ -25℃ storage, valid for one year. Notes 1. This product is for research use only. 2. Please operate with lab coats and disposable gloves, for your safety. Instructions Recommended PCR reaction systems.   Table 1 PCR reaction system Components Volume(μL) Final concentration Template** x - High-Fidelity DNA Polymerase (1 U/μL) 1 1× 2×PCR buffer（Mg2+，dNTPs） 25   Forward Primer(10 μmol/L) 2 0.4 μmol/L Reverse Primer(10 μmol/L) 2 0.4 μmol/L ddH2O Up to 50 - 【Note】: 1) Gently vortex and briefly centrifuge all solutions after thawing. 2) The final concentration of Mg2+ is 2 mM. But it can be varied in a range of 0.2–0.5 mM, if needed. 3) Add 3% DMSO as a PCR additive, which aids in the denaturing of templates with high GC contents. 4) Recommended template dosage (25 μL volume): templates Amplify fragments from 1 kb to 10 kb gDNA 50 ng-200 ng Amplify fragments from 1 kb to 10 kb 10 pg-20 ng cDNA 1-2.5 μL（Do not exceed 10% of the final PCR reaction volume)） Reaction program Two-Step Protocol (priority protocol)                  Three-Step Protocol (regular protocol) Cycle step Temp. Time Cycles   Cycle step Temp. Time Cycles Initial denaturation 98°C 3 min 1 Initial denaturation1 98°C 3 min 1 Denaturation 98°C 10 sec 30-35 Denaturation 98°C 10 sec 30-35 Extension 68°C 30 sec/kb Annealing2 60°C 20 sec Final extension 72°C 5 min 1 Extension3 72°C 30 sec/kb           Final extension 72°C 5 min 1     Annealing Gradient Protocol (complexity template) Cycle step Temp. Time Cycles Initial denaturation1 98°C 3 min 1 Denaturation 98°C 10 sec 15, 1℃/cyc le Gradient annealing2 70-55°C 20 sec Extension3 72°C 30 sec/kb Denaturation 98°C 10 sec 20 Annealing2 55°C 20 sec Extension 72°C 30 sec/kb Final extension 72°C 5 min 1 【Note】: 1. Initial denaturation: We recommend 3 min initial denaturation at 98°C for most templates, recommend 5-10 min for GC-rich template. 2. Annealing: Recommended temperature: 60°C, you can also set a temperature gradient to touch the optimal temperature of index annealing as needed. The recommended annealing time is set to 20 sec, which can be adjusted within  10-30 sec. Annealing time too long may cause the amplification product to spread out on the gel. 3. Extension: Recommended temperature: 72°C. Time: 30 sec/kb, complex templates can be extended to 60 sec/kb depending on the actual situation. *Features under different amplification protocol Protocol Two-step Three-step Gradient annealing Speed fast medium slow Specificity high medium high PCR yield medium high medium Detection rate high medium high  

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Product description This kit is a ready-to-use 2×pre-mixed solution containing High-Fidelity DNA Polymerase, dNTPs, and an optimized buffer system, which contains pre-added electrophoresis indicators. The pre-mix contains pre-added electrophoresis indicator, PCR products can be directly electrophoresed, the amplification products are flat ends. 2×High-Fidelity Fast PCR Master Mix (With Dye) has the advantages of quick and easy, high sensitivity, high specificity, good stability, etc., the reaction system can be added with only the primers and templates. In addition, the product also contains a specific protective agent, so that the premix can still maintain stable activity after repeated freezing and thawing. Components Components No. N132016S N132016M Size 1 mL 5×1 mL Shipping and Storage Dry ice shipping. -15℃ ~ -25℃ storage, valid for one year. Notes 1. This product is for research use only. 2. Please operate with lab coats and disposable gloves, for your safety. Instructions 1. Recommended PCR reaction systems. Table 1 PCR reaction system Components Volume(μL) Final concentration 2×High-Fidelity Fast PCR Master Mix (With Dye) 25 1× Template** x - Forward Primer(10 μmol/L)*** 2 0.4 μmol/L Reverse Primer(10 μmol/L) 2 0.4 μmol/L ddH2O Up to 50 - 【Note】: a. *In 1× premixes containing 2 mM Mg2+ and 200 μM dNTPs. b. **Recommended range 10-200 ng, cDNA sample upload volume range not more than 1/10 of the reaction system, recommended 1-2.5 μL. c. ***The final primer concentration in the PCR reaction system ranges from 0.2-1 μM, and 0.4 μM is recommended. 2. Reaction program Table 2 PCR reaction program Cycle step Temp. Time Cycles Initial denaturation 98℃ 30 sec 1 Denaturation 98℃ 10 sec 30-35 Annealing* 60℃ 5 sec Extension** 72℃ 5-10 sec/kb Final extension 72℃ 2 min 1 【Note】: 1. *Recommended temperature: 60°C, a temperature gradient can be set up to find the optimal temperature for primer annealing. The recommended annealing time is set to 5 sec and can be adjusted from 5-30 sec. Too long annealing time may result in diffuse amplification products on the gel. 2. **Extension time: Recommended 5 sec/kb, can also be extended to 10 sec/kb as needed.  

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