2× High-Fidelity PCR Master Mix (With Dye)

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

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

Components

Volume(μL)

Final concentration

2× High-Fidelity PCR Master Mix (With Dye)*

25

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

Template Type

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  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

 

2× High-Fidelity PCR Master Mix (With Dye)

Product form

SKU: N132014E

$15.00

    • In stock
    • Shipped today? Order within: Jul 11, 2025 17:00:00 -0400

    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

    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

    Components

    Volume(μL)

    Final concentration

    2× High-Fidelity PCR Master Mix (With Dye)*

    25

    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

    Template Type

    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  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

     

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