Description
Product description
This kit is based on the Pyroccus Furiosis DNA Polymerase, genetically engineered. The enzyme has a 5'→3' DNA polymerase activity and a 3'→5' exonuclease activity, and its fidelity is 83 times that of Taq DNA polymerase and 9 times that of ordinary Pfu DNA polymerase. Two monoclonal antibodies at room temperature that inhibit polymerase activity and 3'→5' exonuclease activity were added to the enzyme solution, which can easily perform highly specific Hot Start PCR, greatly improving the detection rate of amplification and the specificity of the product. The addition of an extension factor to the enzyme solution gives the enzyme the ability to amplify long fragments, and the length of the amplification of the fragment of interest can be up to 13 kb. This product is equipped with an optima buffer that makes the enzyme suitable for amplification of complex templates. It generates blunt ends in the amplification products.
Components
|
Components No.
|
Name
|
N132015S
(100 U)
|
N132015M
(500 U)
|
N132015L
(1000 U)
|
|
N132015-A
|
High-Fidelity DNA Polymerase (1 U/μL)
|
100 μL
|
500 μL
|
500 μL×2
|
|
N132015-B
|
2×PCR buffer（with Mg2+，dNTPs）
|
3×1 mL
|
15×1 mL
|
30×1 mL
|
|
N132015-C
|
6×DNA Loading Buffer
|
1 mL
|
6×1 mL
|
12×1 mL
|
Applications
Gene cloning; amplification of complex templates DNA; high-throughput library building.
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
|
Components
|
Volume(μL)
|
Final concentration
|
|
Template**
|
x
|
-
|
|
High-Fidelity DNA Polymerase (1 U/μL)
|
1
|
1×
|
|
2×PCR buffer（Mg2+，dNTPs）
|
25
|
|
|
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) Gently vortex and briefly centrifuge all solutions after thawing.
2) The final concentration of Mg2+ is 2 mM. But it can be varied in a range of 0.2–0.5 mM, if needed.
3) Add 3% DMSO as a PCR additive, which aids in the denaturing of templates with high GC contents.
4) Recommended template dosage (25 μL volume):
|
templates
|
Amplify fragments from 1 kb to 10 kb
|
|
gDNA
|
50 ng-200 ng
|
|
Amplify fragments from 1 kb to 10 kb
|
10 pg-20 ng
|
|
cDNA
|
1-2.5 μL（Do not exceed 10% of the final PCR reaction volume)）
|
Reaction program
Two-Step Protocol (priority protocol) Three-Step Protocol (regular protocol)
|
Cycle step
|
Temp.
|
Time
|
Cycles
|
|
Cycle step
|
Temp.
|
Time
|
Cycles
|
|
Initial denaturation
|
98°C
|
3 min
|
1
|
Initial denaturation1
|
98°C
|
3 min
|
1
|
|
Denaturation
|
98°C
|
10 sec
|
30-35
|
Denaturation
|
98°C
|
10 sec
|
30-35
|
|
Extension
|
68°C
|
30 sec/kb
|
Annealing2
|
60°C
|
20 sec
|
|
Final extension
|
72°C
|
5 min
|
1
|
Extension3
|
72°C
|
30 sec/kb
|
|
|
|
|
|
|
Final extension
|
72°C
|
5 min
|
1
|
Annealing Gradient Protocol (complexity template)
|
Cycle step
|
Temp.
|
Time
|
Cycles
|
|
Initial denaturation1
|
98°C
|
3 min
|
1
|
|
Denaturation
|
98°C
|
10 sec
|
15,
1℃/cyc le
|
|
Gradient annealing2
|
70-55°C
|
20 sec
|
|
Extension3
|
72°C
|
30 sec/kb
|
|
Denaturation
|
98°C
|
10 sec
|
20
|
|
Annealing2
|
55°C
|
20 sec
|
|
Extension
|
72°C
|
30 sec/kb
|
|
Final extension
|
72°C
|
5 min
|
1
|
【Note】:
1. Initial denaturation: We recommend 3 min initial denaturation at 98°C for most templates, recommend 5-10 min for GC-rich template.
2. Annealing: Recommended temperature: 60°C, you can also set a temperature gradient to touch the optimal temperature of index annealing as needed. The recommended annealing time is set to 20 sec, which can be adjusted within 10-30 sec. Annealing time too long may cause the amplification product to spread out on the gel.
3. Extension: Recommended temperature: 72°C. Time: 30 sec/kb, complex templates can be extended to 60 sec/kb depending on the actual situation.
*Features under different amplification protocol
|
Protocol
|
Two-step
|
Three-step
|
Gradient annealing
|
|
Speed
|
fast
|
medium
|
slow
|
|
Specificity
|
high
|
medium
|
high
|
|
PCR yield
|
medium
|
high
|
medium
|
|
Detection rate
|
high
|
medium
|
high
|
