Super Pfx DNA Polymerase (C09-1564-258)

Super Pfx DNA Polymerase is a fast and highly efficient high fidelity DNA polymerase with 5 ′ -3 ′ DNA polymerase activity and 3 ′ -5 ′ exonuclease activity. This enzyme is modified from other high fidelity enzymes, with strong amplification ability, fast amplification speed, and high fidelity, overcoming the shortcomings of poor amplification ability, low yield, and slow amplification speed of ordinary Pfu enzymes, greatly shortening the reaction time. This product can be used for long fragment amplification and other complex template amplification. The 3 'end of the PCR product obtained from amplification does not contain an "A" base and can be directly cloned into a flat end vector. If T/A cloning is required, "A" needs to be added to the end of the PCR product before cloning. This product is suitable for gene cloning, gene directed mutagenesis, SNP amplification experiments, etc.Activity definition:The amount of enzyme required to add 10 nmoL deoxyribonucleotides to acidic insoluble substances within 30 minutes at 74 ℃ is defined as 1 active unit (U).Quality control:After multiple column purifications, SDS-PAGE detected a purity of over 98%: No exogenous nuclease activity detected: Store at room temperature for one month without significant changes in activity.Usage:The following are examples of conventional PCR reaction systems and reaction conditions. In practical operation, corresponding improvements and optimizations should be made based on different templates, primer structures, and target fragment sizes.1. :PCR reaction system:All operations should be carried out on ice. After each group is decomposed and frozen, please mix thoroughly. After use, please put it back at -20 ℃ for storage in a timely manner.Reagent:50 μLReaction system:final concentration:2×Super Pfx Buffer:25 μL:1×:dNTP Mix，10 mM each:1.5-2.5 μL:300-500 μM each:Forward Primer，10 μM:2 μL:0.4 μM:Reverse Primer，10 μM:2 μL:0.4 μM:TemplateDNA appropriate amount:appropriate amount:<:500 ng/50 μL:Super Pfx DNA  Polymerase:0.5-0.75 μL:1-1.5 U/50 μL:ddH2 O:up to 50 μL: :2.  PCR reaction conditions:step:temperature:time: :Pre denaturation:98°C:30 s-3 min: :Degeneration regression:98°C:10-30 s:25-35 cycles:Fire extension:Determine based on primer Tm:15-30 s:25-35 cycles:Final extension:72°C:3-5 kb/min:25-35 cycles:extend:72°C:5 min:Attention:1) Priority should be given to using the three-step method for amplification. If the three-step method cannot amplify the target product or the Tm value of the primer is greater than 68 ° C, please try the two-step method.2) Denaturation: Pre denaturation of simple templates at 98 ℃ for 30 seconds to 1 minute. For complex templates, the pre denaturation time can be extended to 3 minutes.3) Annealing: In general experiments, the annealing temperature is 3-5 ℃ lower than the Tm value of the primer. If the ideal amplification efficiency cannot be achieved, the annealing temperature should be gradually changed for optimization: When non-specific reactions occur, increase the annealing temperature appropriately.4) Extension: The extension time should be set according to the length of the amplified fragment and the complexity of the template. The amplification efficiency of this product is 3-5 kb/min, and it is recommended to 2-4 kb/min for long fragments and high complexity templates.5) Cycle count: The number of cycles can be set based on the downstream application of the amplification product. If there are too few cycles, insufficient amplification, or too many cycles, the probability of mismatch will increase. Therefore, while ensuring product yield, the number of cycles should be minimized as much as possible.