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. The mix includes two monoclonal antibodies that inhibit polymerase activity and 3’→5’exonuclease activity at room temperature, enabling highly specific Hot Start PCR. The addition of elongation factors allows for long-fragment amplification, with target fragments up to 13 kb. This enzyme exhibits 5’→3’ DNA polymerase activity and 3’→5’ exonuclease activity, with a fidelity 83 times that of Taq DNA polymerase and 9 times that of ordinary Pfu DNA polymerase. It is suitable for amplifying complex templates, producing blunt-end PCR products.
The kit offers advantages such as simplicity, high sensitivity, strong specificity, and excellent stability. The reaction system only requires the addition of primers and template, and a two-step PCR protocol can be used to simplify the procedure and save time. The product includes electrophoresis tracking dye, allowing direct loading of PCR products for gel analysis. Additionally, it contains a special protective agent that ensures stable activity even after repeated freeze-thaw cycles.
Components
|
Components
|
N132014S
|
N132014M
|
N132014L
|
|
2× High-Fidelity PCR Master Mix (With Dye)
|
1 mL
|
5×1 mL
|
100×1 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
Recommended PCR reaction systems.
Table 1 PCR reaction system
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Components
|
Volume(μL）
|
Final concentration
|
|
2× High-Fidelity 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】:
1) *The 1× final concentration of the mix contains 1 U/50 μL of polymerase, 2 mmol/L Mg2+, and 200 μmol/L dNTPs.
2) **The recommended final concentration of primers in the PCR reaction is 0.2-1 μmol/L, with 0.4 μmol/L preferred.
3) ***Recommended template amounts for a 25 μL reaction system:
Table 2 Recommended template amounts
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Template Type
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Fragment Size (1 kb-10 kb)
|
|
Genomic DNA
|
50 ng-200 ng
|
|
Plasmid or Viral DNA
|
10 pg-20 ng
|
|
cDNA
|
1-2.5 μL (not exceeding 1/10 of the total PCR volume)
|
Reaction program.
1）Two-Step Protocol (Preferred):
|
Cycle step
|
Temp.
|
Time
|
Cycles
|
|
Initial denaturation
|
98℃
|
3 min
|
1
|
|
Denaturation
|
98℃
|
10 sec
|
30-35
|
|
Extension
|
68℃
|
30 sec/kb
|
|
Final extension
|
72℃
|
5 min
|
1
|
2）Three-Step Protocol (Conventional):
|
Cycle step
|
Temp.
|
Time
|
Cycles
|
|
Initial denaturation*
|
98℃
|
3 min
|
1
|
|
Denaturation
|
98℃
|
10 sec
|
30-35
|
|
Annealing**
|
60℃
|
20 sec
|
|
Extension***
|
72℃
|
30 sec/kb
|
|
Final extension
|
72℃
|
5 min
|
1
|
【Note】:
a. * Initial Denaturation: Recommended at 98°C for 3 min (5-10 min for high-GC templates).
b. **Annealing: Recommended at 60°C for 20 sec (adjustable between 10-30 sec). Prolonged annealing may cause smearing on gels.
c. ***Extension: Recommended at 72°C for 30 sec/kb (up to 60 sec/kb for complex templates).
3）Gradient Annealing Protocol (Recommended for Difficult Targets):
|
Cycle step
|
Temp.
|
Time
|
Cycles
|
|
Initial denaturation
|
98℃
|
3 min
|
1
|
|
Denaturation
|
98℃
|
10 sec
|
15 (decrease by 1° C per cycle)
|
|
Gradient Annealing
|
70-55°C
|
20 sec
|
|
Extension
|
72℃
|
30 sec/kb
|
|
Denaturation
|
98°C
|
10 sec
|
20
|
|
Annealing
|
55°C
|
20 sec
|
|
Extension
|
72°C
|
30 sec/kb
|
|
Final extension
|
72℃
|
5 min
|
1
|
4）Characteristics of Different PCR Protocols:
|
Protocol Type
|
Speed
|
Specificity
|
PCR Yield
|
Detection Rate
|
|
Two-Step
|
Fastest
|
High
|
Medium
|
High
|
|
Three-Step
|
Medium
|
Medium
|
Highest
|
Medium
|
|
Gradient Annealing
|
Slow
|
High
|
Medium
|
High
|
