Description
Pfu DNA Polymerase is a DNA polymerase derived from thermophilic archaea. In addition to its 5'-3' DNA polymerase activity, this enzyme also has strong 3'-5' exonuclease activity and enhances PCR amplification. Fidelity. Compared to the Taq enzyme, pfu polymerase is more thermostable and more resistant to higher denaturation temperatures. The fidelity of Pfu is 10-15 times that of Taq DNA polymerase. The extension speed was 0.5 kb/min. The 3' end of the PCR product has no base A and is a smooth end, and is cloned by a smooth end cloning method. 1 unit (U) pfu DNA Polymerase activity is defined as the requirement for the incorporation of 10 nmol deoxynucleotides into acid-insoluble materials using activated salmon salmon sperm DNA as a template/primer at 74 ° C for 30 minutes. The amount of enzyme.
Product number and specifications :
| ZK06035 |
pfu DNA Polymerase
|
500Ux 1 |
| ZK06036 |
pfu DNA Polymerase
|
500U x 5 |
Storage conditions: -20℃.
Concentration: 2.5U/ul
Enzyme storage buffer:50 mM Tris-HCl (pH 8.0); 0.1 mM EDTA; 1 mM DTT; 50 mM KCl ; Stabilizers; 50% glycerol.
Scope of application: Amplification of high-fidelity DNA fragments and smoothing of DNA fragments.
Instructions for use:
- Prepare the reaction system in a 0.2 ml PCR tube according to the following table :
| Ingredient |
20ul |
50ul |
Final concentration |
| Template |
0.04-0.2 μg |
0.1-0.5 μg |
as you wish |
| Primer 1（10 mM） |
0.4 μl |
1 |
0.2 μM |
| Primer 2（10 mM） |
0.4 μl |
1 |
0.2 μM |
| 10×Taq Buffer |
2 μl |
5 |
1x |
| dNTP Mixture(10 mM) |
0.4 μl |
1 |
200 μM each |
| Taq (5 U/ml) |
0.4 μl |
1 |
0.05U/μl |
| ddH2O |
Up to 20ul |
Up to 50ul |
|
Note 1: If you need to change the Mg2+ concentration, adjust according to the following table.
| Final concentration of Mg2+ in the PCR reaction system |
2mM |
2.5mM |
3mM |
4mM |
| 25 mM MgSO4 in 20 μl reaction system |
0 |
0.4ul |
0.8ul |
1.6ul |
| 25 mM MgSO4 in 50 μl reaction system |
0 |
1ul |
2ul |
4ul |
Note 2: When preparing the reaction solution, please add pfu last because the enzyme has strong 3'-5' exonuclease activity and may degrade the primer.
- After mixing, centrifuge the solution and collect the reaction solution to the bottom of the tube.
- If the PCR instrument is not heated by a hot lid, add 25 μl of mineral oil.
- Perform the following procedure on the PCR machine:
| Step |
Temperature |
Time |
Cycle |
| Initial Denaturation |
94℃ |
3-5min |
1 |
| Transsexual |
94℃ |
30sec |
25-40 |
| Annealing |
50-60℃ |
30sec |
| Extend |
72℃ |
0.5kb/min |
| Final extension |
72℃ |
5min |
1 |
Note: The PCR reaction conditions vary depending on the structural conditions of the template, primers, and the like. In actual practice, the optimal reaction conditions (temperature, time, etc.) should be set according to the specific conditions such as the size of the template, the size of the target fragment, the length of the base, and the length of the primer.
Precautions:
- The pfu enzyme has 3'-5' exonuclease activity, so the extension speed of the pfu enzyme is much lower than that of the Taq enzyme. The extension time should be set according to the length of the amplification product. In general, the extension of the pfu enzyme is extended. The rate is 0.5 kb per minute; at the same time, the exonuclease activity of the 3'-5' of the pfu enzyme may degrade the primer, so the pfu enzyme should be finally added to the reaction system and the PCR reaction is immediately performed.
- The thermal stability of pfu enzyme is better than that of Taq enzyme. For template with high GC content, the denaturation temperature can be increased to 98 °C, which has no effect on the activity of pfu enzyme.
