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
Elongation capability
G-HiFi™ DNA Polymerase’s high processability enables reliable amplification of λDNA up to 40 kb in length (M: DM5100).
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
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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
What are the differences between SMO-HiFi™ (TF1000) and G-HiFi™ (TF3000)?
SMO-HiFi™ (TF1000) and G-HiFi™ (TF3000) contain different high fidelity DNA polymerases. SMO-HiFi™ (TF1000) and G-HiFi™(TF3000) DNA polymerases are suitable for GC-rich templates that are difficult to amplify. Moreover G-HiFi™ (TF3000) is blended with elongation enhancer to perform quick and accurate PCR.
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 |
Templatedenature | 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
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
[NS1000] FluoroVue™ Nucleic Acid Gel Stain (10,000X), 500 μl
[DS1000] FluoroStain™ DNA Fluorescent Staining Dye (Green, 10,000X), 500 μl
[DL5000] FluoroDye™ DNA Fluorescent Loading Dye (Green, 6X), 1 ml
Blue-light illuminator
[VE0100] B-BOX™ Blue Light LED Epi-illuminator
Ligation
Blund-end PCR amplicons can directly ligate with PCR cloning vector.
[CV1100] GetClone™ PCR Cloning Vector II, 20 Rxn
Transformation
Prepare competent cells with high efficiency and transform with time-saving protocol.
[CK1000] Champion™ E. coli Transformation Kit
Colony PCR
Analyze colonies with PCR master mix to save preparation time.
[TP1100] ExcelTaq™ 5X PCR Master Mix, 200 Rxn
[TP1200] ExcelTaq™ 5X PCR Master Dye Mix, 200 Rxn
