2× MegaPfu Premix HYA448
2× MegaPfu Premix is a ready-to-use high-fidelity PCR premix containing MegaPfu DNA polymerase, buffer, dNTP Mixture, and stabilizer. When using, you only need to add templates and primers to the product solution to perform PCR reactions, which greatly simplifies the operation process and reduces the risk of contamination during PCR operations. The MegaPfu DNA Polymerase used in this product has DNA polymerase activity in the 5'→3' direction and DNA exonuclease activity in the 3'→5' direction. It can correct base mismatches generated during DNA amplification and is a high-fidelity PCR fast reaction enzyme. The PCR product amplified using this product has a blunt end. If TA cloning is performed, it needs to be treated with A before being connected to the T vector.
Components
|
Components |
Size-1 |
Size-2 |
Size-3 |
|
2× MegaPfu Premix |
1 mL |
1 mL×5 |
1 mL×15 |
Storage
Store at -20°C.
Instructions
1. Reaction System
|
Components |
Volume |
|
2× MegaPfu Premix |
25uL |
|
Forward Primer (10 μM) |
2 µL |
|
Reverse Primer (10 μM) |
2 µL |
|
DNA Template* |
Variable |
|
ddH2O |
Up to 50 µL |
*Different sources or types of templates require different reaction concentrations. The following table shows the recommended template usage for a 50 µl reaction system:
|
DNA |
Input Amount |
|
Plants, animals and human gDNA |
0.1-1 µg |
|
E.coli gDNA |
10-100 ng |
|
cDNA |
1-5 μL (no more than 1/10 of the total PCR reaction volume) |
|
Plasmid DNA |
0.1-10 ng |
|
λDNA |
0.5-10 ng |
2. Reaction Program
|
Step |
Temperature |
Time |
Cycles |
|
Initial Denaturation |
95 ℃ |
2 min |
1 |
|
Denaturation |
95 ℃ |
15 s |
30-35 |
|
Annealing |
56 ℃ |
15 s |
|
|
Extension |
72℃ |
30 s/kb |
|
|
Final Extension |
72℃ |
5 min |
1 |
|
Hold |
4-12 ℃ |
- |
1 |
Note:The annealing temperature needs to be adjusted according to the actual GC content of the primer and template; if necessary, a temperature gradient can be set to find the optimal temperature for primer-template binding.