Description
Product description
This kit is a ready-to-use 2×pre-mixed solution containing High-Fidelity DNA Polymerase, dNTPs, and an optimized buffer system, which contains pre-added electrophoresis indicators. The pre-mix contains pre-added electrophoresis indicator, PCR products can be directly electrophoresed, the amplification products are flat ends. 2×High-Fidelity Fast PCR Master Mix (With Dye) has the advantages of quick and easy, high sensitivity, high specificity, good stability, etc., the reaction system can be added with only the primers and templates. In addition, the product also contains a specific protective agent, so that the premix can still maintain stable activity after repeated freezing and thawing.
Components
|
Components No.
|
N132016S
|
N132016M
|
|
Size
|
1 mL
|
5×1 mL
|
Shipping and Storage
Dry ice shipping. -15℃ ~ -25℃ storage, valid for one year.
Notes
1. This product is for research use only.
2. Please operate with lab coats and disposable gloves, for your safety.
Instructions
1. Recommended PCR reaction systems.
Table 1 PCR reaction system
|
Components
|
Volume(μL)
|
Final concentration
|
|
2×High-Fidelity Fast PCR Master Mix (With Dye)
|
25
|
1×
|
|
Template**
|
x
|
-
|
|
Forward Primer(10 μmol/L)***
|
2
|
0.4 μmol/L
|
|
Reverse Primer(10 μmol/L)
|
2
|
0.4 μmol/L
|
|
ddH2O
|
Up to 50
|
-
|
【Note】:
a. *In 1× premixes containing 2 mM Mg2+ and 200 μM dNTPs.
b. **Recommended range 10-200 ng, cDNA sample upload volume range not more than 1/10 of the reaction system, recommended 1-2.5 μL.
c. ***The final primer concentration in the PCR reaction system ranges from 0.2-1 μM, and 0.4 μM is recommended.
2. Reaction program
Table 2 PCR reaction program
|
Cycle step
|
Temp.
|
Time
|
Cycles
|
|
Initial denaturation
|
98℃
|
30 sec
|
1
|
|
Denaturation
|
98℃
|
10 sec
|
30-35
|
|
Annealing*
|
60℃
|
5 sec
|
|
Extension**
|
72℃
|
5-10 sec/kb
|
|
Final extension
|
72℃
|
2 min
|
1
|
【Note】:
1. *Recommended temperature: 60°C, a temperature gradient can be set up to find the optimal temperature for primer annealing. The recommended annealing time is set to 5 sec and can be adjusted from 5-30 sec. Too long annealing time may result in diffuse amplification products on the gel.
2. **Extension time: Recommended 5 sec/kb, can also be extended to 10 sec/kb as needed.
