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Products Introduction
Super Kfx DNA Polymerase is a high-fidelity DNA polymerase with 5′-3′ DNA polymerase activity and 3′-5′ exonuclease activity for fast and efficient amplification. The polymerase has 5′-3′ DNA polymerase activity and 3′-5′ exonuclease activity. The enzyme has been modified from other high-fidelity enzymes by adding unique extension factors and specific promoter factors, which greatly improves the amplification ability, overcomes the defects of ordinary Pfu enzyme's poor amplification ability, low yield, and slow amplification speed, and shortens the reaction time. This product can be used for the amplification of ordinary fragments, long fragments and other complex templates, the 3′ end of the amplified PCR product does not have the "A" base, if you need to carry out T/A cloning, you need to add the "A" at the end of the PCR product and then clone it. This product is suitable for gene cloning, second generation library amplification, targeted gene mutation, SNP amplification experiments.
Active Definition
The amount of enzyme required to incorporate 10 nmoL of deoxyribonucleotide into an acid-insoluble substance is defined as 1 active unit (U) at 74°C for 30 min.
Quality control
After several column purifications, the purity of the product was greater than 98% by SDS-PAGE; no exogenous nuclease activity was detected; and the product was stored at room temperature for one month without any obvious activity change.
Usage
The following are examples of conventional PCR reaction systems and conditions, which should be improved and optimized according to the template, primer structure and fragment size.
1. PCR reaction system
All operations should be carried out on ice, please mix the components thoroughly after thawing, after use, please promptly return to -20 ℃ for storag
2. PCR reaction system
Take note of
1）Priority is given to three-step amplification; if the three-step method fails to amplify the target product or if the primer Tm value is greater than 68°C, try the two-step method.
2）Denaturation: pre-denaturation of simple templates 98°C, 30s-1min, for complex templates, the pre-denaturation time can be extended to 3min.
3）Annealing: In general, the annealing temperature is 3-5℃ lower than the Tm value of the primers. If the desired amplification efficiency cannot be obtained, the annealing temperature should be changed in a gradient to optimize the results; if a non-specific reaction occurs, the annealing temperature should be increased appropriately.
4）Extension: The extension time should be set according to the length of the amplified fragments and the complexity of the template, the amplification efficiency of this product is 4-6kb/min, for long fragments and templates with high complexity it is recommended that 2-4kb/min.
5）Cycling times: the number of cycles can be set according to the downstream application of the amplification product. If the number of cycles is too small, the amplification amount will be insufficient, and if the number of cycles is too large, the chance of mismatch will be increased, so the number of cycles should be minimized under the premise of guaranteeing the yield of the product.
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