Home Page/Product Center

Hieff Canace® Gold High-Fidelity DNA Polymerase

Product SKU

Specifications

Price

10148ES10

10 U

$15.90

10148ES60

100 U

$105.46

10148ES76

500 U

$367.94

10148ES80

1000 U

$707.62

Product Description

Hieff Canace® Gold High-Fidelity DNA Polymerase obtained by genetic engineering technology on the basis of Pyrococcus Furiosis DNA Polymerase. Gold High-Fidelity DNA Polymerase possesses 5’-3’ polymerase activity and 5’-3’ exonuclease activity, and the fidelity of this enzyme is 52-fold of Taq DNA Polymerase and 6-fold of conventional Pfu. The hot-start factors added to this enzyme greatly improve the detection rate and specificity of the product.

Gold High-Fidelity DNA Polymerase combined with an elongation factor to successfully amplify extremely long PCR amplicons, which amplification of targets up to 10 kb, and extension time to 15 sec/kb. The optimized buffer and PCR enhancer are supplied with the enzyme, which performs extremely well on complex templates. The amplification will generate blunt-ended products.

Product Information

Component Number

Components

Product Number/ specification
10148ES10 (10 U)

Product Number/ specification
10148ES60 (100 U)

Product Number/ specification
10148ES76 (500 U)

Product Number/ specification
10148ES80 (1000 U)

10148-A

Hieff Canace® Gold High-Fidelity DNA Polymerase (2 U/μL)

5 μL

50 μL

250 μL

250 μL×2

10148-B

2×Canace® Gold PCR buffer(with Mg2+,dNTPs)

300 μL

1 mL×3

1 mL×15

1 mL×30

Applications

Gene cloning; complex DNA template amplification; high-throughput library construction.

Unit definition

One unit (U) is defined as the amount of enzyme that incorporates 10 nmol of whole dNTPs into acid-insoluble products in 30 minutes at 74℃ with activated salmon sperm DNA as the template / primer.

Quality Control

Exonuclease Activity: In a 20μl system, Incubation of 10 U of this enzyme with 1 μg λ-Hind III at 37℃ for 4 hours results in no change on electrophoretic bands.

Endonuclease Activity: In a 20μl system, Incubation of 10 U of this enzyme with 1 μg λDNA for 4 hours at 37℃ results in no change on electrophoretic bands.

Residual E.Coli gDNA Test: In a 50μl system with 2U of this enzyme, amplify E.coli 16s rDNA gene with sterile ddH2O was used as template. After 30 cycles, PCR products were detected by 1% agarose gel electrophoresis. None DNA band detected after EB staining.

Storage and Stability

Transport using ice bag.

The product can be stored at -20℃ for 24 months.

Important Notes

For your safety and health, a lab coat and disposable gloves must be worn.

Recommended PCR System (on ice during the experiment)

Components

Volume

Final Concentration

ddH2O

to 50 μL

-

2×Canace® Gold PCR buffer (with Mg2+,dNTPs)

25 μL

Template DNA

X μL

-

Primer 1 (10 μM)

2.5 μL

0.5 μM

Primer 2 (10 μM)

2.5 μL

0.5 μM

Hieff Canace® Gold High-Fidelity DNA Polymerase (2 U/μL)

0.5 μL

1 U/50 μL

Before use, dissolve all components by incubating on ice and gently flipping the tube. Mix the solution thoroughly every time before pipetting.

The final concentration of polymerase is usually 1 U/50 μL. It can be optimized between 0.5-2 U/50 μL, please do not exceed 2 U/50 μL.

In order to prevent the polymerase from degrading the primer due to the 3'→5' exonuclease activity, it is recommended that the polymerase be added to the reaction system in the last step.

The final concentration of Mg2+ is usually 1.5 mM, and if necessary, it can be adjusted between 0.2-0.5 mM using 50 mM MgCl2.

For high GC templates, adding DMSO at a final concentration of 3% to the system may be beneficial to amplification.

Recommended templates (In a 50 µl system):

Templates

Input Template DNA1 kb-10 kb

Genomic DNA

50 ng-200 ng

Plasmid or Virus DNA

10 pg-20 ng

cDNA

1-5 μL (≤ 1/10 of the total volume of PCR system)

PCR amplification program: There are the following three programs to choose from, the two-step program is preferred.

2-step PCR Program (Preferentially recommended):

Steps

Temperature

Time

Cycles

Pre-denaturation

98℃

3 min

1

Denatu tion

98℃

10 sec

30-35

Extension

68℃

30 sec/kb

30-35

Final Extension

72℃

5 min

1

3-step PCR Program (Standard program):

Steps

Temperature

Time

Cycles

Pre-denaturation

98℃

3 min

1

Denaturation

98℃

10 sec

30-35

Annealing

60℃

20 sec

30-35

Extension

72℃

30 sec/kb

30-35

Final Extension

72℃

5 min

1

Touch Down PCR Program (Complex templates):

Steps

Temperature

Time

Cycles

Pre-denaturation

98℃

3 min

1

Denaturation

98℃

10 sec

15. Reduce by 1℃ per cycle

Gradient annealing

70-55℃

20 sec

15. Reduce by 1℃ per cycle

Extension

72℃

30 sec/kb

15. Reduce by 1℃ per cycle

Den uration

98℃

20 sec

20

Normal annealing

55℃

20 sec

20

Extension

72℃

30 sec/kb

20

Final Extension

72℃

5 min

1

* Characteristics of different amplification procedures:

Program type

2-step PCR Program

3-step PCR Program

Gradient annealing

Speed

The fastest

Medium

Slow

Specificity

High

Medium

High

Yield

Medium

The highest

Medium

Extension

72℃

30 sec/kb

30-35

Detection rate

High

Medium

High

Note:

For pre-denaturation, pre-denaturation at 98℃ for 3 min is suitable for most amplification. However, it could be prolonged to 5-10 min for high GC template.

For annealing, the recommended temperature is 60℃, if necessary, set gradient annealing temperature to find out the optimal one.
In addition, the time is 20 sec, which can be adjusted within 10-30 sec. Too long annealing time may cause the amplified product to become diffuse on the gel.

For denaturation, denaturation at 72℃ for 30 sec/kb is suitable for most amplification., while for complex templates, the extension time is recommended to prolong to 60 sec/kb depending on the situation.

PCR products, please store the PCR products at -20℃ to prevent the enzyme from degrading the amplification product. The amplified product is blunt-ended.

15221439726

15221439726