[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. 


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


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 


 

[TF3000] G-HiFi™ DNA Polymerase

Manual

Manual_TF3000_G-HiFi™ DNA Polymerase

SDS

SDS_TF3000


 


 

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.

 

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.