[TF3000] G-HiFi™ DNA Polymerase, (1 U/μl, 100 U)

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Description 

The G-HiFi™ DNA Polymerase is a new genetically modified, recombinant DNA polymerase suitable for GC-rich templates that are difficult to amplify. The fidelity of G-HiFi™ DNA Polymerase is 70 times higher than that of Taq DNA polymerase. The high extension rate of G-HiFi™ DNA Polymerase is achieved by blending the DNA polymerase with an elongation enhancer. The optimized 5X G-HiFi™ Buffer includes special ingredients that suppress non-specific amplification as well as plateau effect produced by conventional PCR. With the optimized 5X G-HiFi™ Buffer, G-HiFi™ DNA Polymerase is capable to amplify most templates, such as longer targets (up to 40 kb from lambda DNA) and that contain GC-rich sequences. 


Additional Format

The G-HiFi™ DNA Polymerase is also available in a master mix format. The G-HiFi™ 2X PCR Master Mix (Cat. No. TF3100) is a ready-to-use mixture for amplifying targeted DNA fragments. It is designed for virtually all PCR applications. The mixture contains all essential ingredients for PCR with the exception of primers and template. This not only saves valuable time in the laboratory, but also reduces pipetting and reagent handling errors.  


Features

  • 5’→3’ DNA polymerase activity 

  • 3’→5’ exonuclease (proofreading) activity 

  • High reaction rate: 7 seconds/kb 

  • High fidelity: 70 times higher than Taq polymerase 

  • Generates blunt end amplicons  

  • Vast elongation capability (up to 40 kb) 

  • Thermo-stable for more than 10 hrs at 95°C 


Storage

[TF3000] G-HiFi™ DNA Polymerase

-20°C for 24 months

[TF3100] G-HiFi™ 2X PCR Master Mix

-20°C for 6 months

Odoo - Sample 1 for three columns

Elongation capability

G-HiFi™ DNA Polymerase’s high processability enables reliable amplification of λDNA up to 40 kb in length (M: DM5100). 

Odoo - Sample 2 for three columns

Sensitivity

G-HiFi™ DNA Polymerase performs higher sensitivity for high GC content templates (GC: 71%) compare to high fidelity DNA Polymerase from Brand A (M: DM2000).

 

[TF3000] G-HiFi™ DNA Polymerase

Contents

Component

Volume

G-HiFi™ DNA Polymerase (1 U/μl)

100 μl

5X G-HiFi™ Buffer

1200 μl

dNTPs Mix (2 mM each)

600 μl


Storage Buffer

50 mM Tris-HCl (pH 8.0), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, stabilizer, 50% (v/v) glycerol


Unit Definition

One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 74°C. 


Storage

-20°C for 24 months 


[TF3100] G-HiFi™ 2X PCR Master Mix

Contents

Component

Volume

G-HiFi™ 2X PCR Master Mix

1.25 ml

6X DNA Loading Dye (Blue)

1 ml


Storage

-20°C for 6 months 

 

[TF3000] G-HiFi™ DNA Polymerase

Manual

Manual_TF3000_G-HiFi™ DNA Polymerase

MSDS

MSDS_TF3000


[TF3100] G-HiFi™ 2X PCR Master Mix

Manual

Manual_TF3100_G-HiFi™ 2X Master Mix

MSDS

MSDS_TF3100

 


 

Recommended PCR Condition


[TF3000] G-HiFi™ DNA Polymerase

Template

1 – 150 ng

Forward primer

0.1 – 0.5 µM*

Reverse primer

0.1 – 0.5 µM*

5X G-HiFi Buffer

10 µl

dNTPs (2 mM each)

5 µl

G-HiFi DNA Polymerase

0.5 – 1 unit**

H2O

to 50 µl

Total volume

50 µl

*When amplifying products  10 kb in length, use primers at a final concentration of 0.1 μM each.

** When amplifying products  2 kb in length, use 0.5 unit of G-HiFi DNA Polymerase.

 

 [TF3100] G-HiFi™ 2X PCR Master Mix

Template

1 – 150 ng

Forward primer

0.1 – 0.5 µM*

Reverse primer

0.1 – 0.5 µM*

G-HiFi™ 2X PCR Master Mix

25 µl

H2O

to 50 µl

Total volume

50 µl

*When amplifying products  10 kb in length, use primers at a final concentration of 0.1 μM each.

 

Recommended PCR Program

For  10 kb products

Select primers with a Tm value of 55°C. 20- to 25-mer primers are suitable, or those greater than 25-mer in length may provide optimal results.

Steps

Temp.

Time

Cycles

Template denature

98°C

2 min

1

Denature

98°C

10 sec

25-40

Annealing

50-68°C

15 sec

Extension

68°C

10-30 sec/kb

Final extension

68°C

1 min

1

 *Optimal PCR condition varies according to primers’ thermodynamic properties.


For  10 kb products

Select primers with a Tm value of  65°C. 25- to 35-mer primers are suitable. Avoid high GC-content at the 3' end of each primer.

Steps

Temp.

Time

Cycles

Denature

98°C

10 sec

25-40

Extension

68°C

10-30 sec/kb

 

For GC-rich templates:

Steps

Temp.

Time

Cycles

Template denature

98°C

2 min

1

Denature

98°C

10 sec

25-40

Extension

68°C

10-30 sec/kb





AFEAP cloning: a precise and efficient method for large DNA sequence assembly

Fanli Zeng,1 Jinping Zang,1 Suhua Zhang,2 Zhimin Hao,1 Jingao Dong,corresponding author1 and Yibin Lincorresponding author3

BMC Biotechnol. 2017; 17: 81. Published online 2017 Nov 14. doi: 10.1186/s12896-017-0394-x

PMCID: PMC5686892


Odoo - Sample 2 for three columns

Gel electrophoresis

Staining amplicons with safe fluorescent dyes, following by observation under blue-light illuminator to minimize damage of DNA amplicons and maximize successful cloning efficiency.   

Safe fluorescent dyes

Blue-light illuminator

Odoo - Sample 3 for three columns

Ligation

Blund-end PCR amplicons can directly ligate with PCR cloning vector.  

Odoo - Sample 3 for three columns

Transformation

Prepare competent cells with high efficiency and transform with time-saving protocol.

Odoo - Sample 3 for three columns

Colony PCR

Analyze colonies with PCR master mix to save preparation time.