2×HiFi PCR Mix for NGS
This product is a premixed system composed of hot start enzymes, PCR Buffer, dNTPs, Mg2+, PCR stabilizers and enhancers, etc. It has the characteristics of high fidelity, high elongation, and low bias. It has a balanced amplification efficiency for complex DNA templates (such as high GC content templates), and is particularly suitable for the amplification of multiple PCR in second-generation library construction.
The high-efficiency hot start enzyme contained in this product has no polymerase activity at room temperature, effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimers at room temperature. The combination of a unique buffer system and hot start enzymes significantly improves the amplification efficiency of PCR, with a wider amplification range. This product effectively improves the amplification efficiency of high GC or high AT regions in the genome, reduces amplification preference, and improves sequencing coverage.
Component 1mL 5mL
two × HiFi PCR Mix 1mL 5 × 1mL
DdH2O 1mL 5 × 1mL
Storage: -20 ℃, avoid repeated freeze-thaw.
This product is not suitable for primers with modifications.
Before use, please gently mix it upside down and avoid foaming as much as possible. After briefly centrifuging, use it.
Avoid repeated freeze-thaw, as repeated freeze-thaw may cause a decrease in product performance. If frequent use is required in the short term, it can be stored at 2-8 ℃.
The following examples are the conventional PCR reaction system and reaction conditions. In practical operation, corresponding improvements and optimizations should be made based on different templates, primer structures, and target fragment sizes.
Preparation of PCR reaction system
Ingredient dosage
two × HiFi PCR Mix 25 μ L
Primer Pool 0.1-0.3 μ M
DNA or cfDNA 5ng~100ng
DdH2O to 50 μ L
Attention: The primer concentration should be between 0.1 and 0.3 at the final concentration μ M serves as a reference for setting the range.
Set up PCR reaction program
Step Temperature Time Cycles
Pre denaturation at 95 ℃ for 2 minutes 1
Denaturation at 98 ℃ for 20s, 30-40
Annealing extension at 65 ℃ for 90 seconds
Final extension 72 ℃ for 5 minutes 1
Attention:
In general experiments, the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and when ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When a non-specific reaction occurs, increase the annealing temperature to optimize the reaction conditions.
The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatch will increase, and the non-specific background will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible.
