| Product Description | This product is a premixed system composed of Pfu DNA Polymerase, Mg2+, dNTPs, PCR stabilizers and enhancers, with a concentration of 2 ×. Pfu DNA Polymerase exhibits 5 ′ -3 ′ DNA polymerase activity and 3 ′ -5 ′ exonuclease activity, thus possessing error correction ability during DNA amplification. Compared with Taq DNA Polymerase, it has high fidelity (6-8 times that of Taq enzyme) and better thermal stability. The pre prepared PCR mixture makes the operation simpler and faster, minimizing human error and contamination to the greatest extent possible. The original MasterMix formula makes the entire reaction system very stable and has good repeatability. This product has been added with a dye (blue), and can be directly subjected to electrophoresis detection after the reaction is completed. The Pfu DNA polymerase contained in this product has the characteristics of low mismatch rate and high temperature resistance, making it suitable for gene cloning, gene directed mutagenesis, SNP and end effector complement reactions.
| P665594 |
Component |
1 mL |
5 mL |
Storage |
| P665594A |
2×Pfu MasterMix (Dye) |
1 mL |
5×1 mL |
-20℃. Avoid freeze/thaw cycle. |
| P665594B |
ddH2O |
1 mL |
5×1 mL |
-20℃. Avoid freeze/thaw cycle. |
Notes: 2×Pfu MasterMix contains Pfu DNA Polymerase, 3mM MgCl2 and 400µM each dNTP.
|
Quality control:
After testing, there was no exogenous nuclease activity; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes from multiple genomes; Storage at 2-8 ℃ for three months showed no significant change in activity.
Usage:
The following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment using human genomic DNA as a template. In practical operation, corresponding improvements and optimizations should be made based on the template, primer structure, and target fragment size.
1. PCR reaction system
| Reagent |
50 μlReaction system |
Final concentration |
| 2×Taq MasterMix（Dye） |
25 μL |
1× |
| Forward Primer，10 µM |
2 μL |
0.4 μM |
| Reverse Primer，10 µM |
2 μl |
0.4 μM |
| Template DNA |
<0.5 μg |
<0.5 μg/50 μL |
| ddH2O |
up to 50 μL |
/ | |
Attention: When amplifying with Pfu enzyme, the purity of the primers is required to be high, and the length of the primers is greater than 18 bases. The primer concentration should be based on the final concentration of 0.1-1.0 μ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system. 2. PCR reaction conditions
| Step |
Temperature |
Time |
/ |
| Pre denaturation |
94℃ |
2 min |
/ |
| Denaturation |
94℃ |
30 s |
25-35 cycles |
| Anneal |
55-65℃ |
30 s |
25-35 cycles |
| Extend |
72℃ |
60 s |
25-35 cycles |
| Finally extended |
72℃ |
5 min |
/ |
|
Attention:
1) The thermal stability of Pfu enzyme is better than that of Taq enzyme. For templates with high GC content, the denaturation temperature can be increased to 98 ℃ without affecting the activity of Pfu enzyme.
2) In general experiments, if the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.
3) Pfu enzyme has 3 '-5' exonuclease activity, so the extension rate of Pfu enzyme during amplification is much lower than that of Taq enzyme. The extension time is set according to the size of the amplified fragment, and the amplification extension rate of this product is 1 kb/min.
4) 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 mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible.
5) This product has 3 '-5' exonuclease activity. The PCR product is a flat end and cannot be directly used for T/A cloning. If T/A cloning is required, "A" needs to be added at its end or cloned using a flat end vector.
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