2× High-Fidelity PCR Master Mix (With Dye)

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