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I-5™ Hi-Fi DNA Polymerase

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250 Units, 2.5 U/μl

General Description 
I-5 Hi-Fi DNA polymerase is an ultra-fast and high-fidelity DNA polymerase. It provides robust amplification of from different templates including plasmids, BACS, genomic DNA, and lambda DNA. It allows for amplification of up to 8kb with human genomic DNA and up to 21kb with lambda DNA. It has an extension speed of 1 kb / 10-15 seconds depending on template type. This allows the user to save time by speeding up PCR reactions and provides higher fidelity than Taq or Pfu.

• Robust – maximal success and minimal optimization needed
• Fidelity – 50X greater than Taq
• High Speed –10X faster than Pfu
• High Yield
• Versatile – best for long or difficult templates

E. coli

Long or Difficult Amplification
High-Throughput PCR

Supplied with 
5X Reaction Buffer (1ml), 50mM MgCl2 (1ml)

Supplied in (buffer description)
20mM Tris-HCl, 100mM KCl, 1mM DTT, 0.1mM EDTA, 200ug/ml BSA, 50% glycerol, 1X stabilizer, pH 7.4 @ 25 C

Storage Condition 

Unit Definition 
One unit is the amount of enzyme that incorporates 5 nmol of dNTPs into acid insoluble material in 15 minutes at 72°C.

Frey, B. and Suppman, B. (1995). BioChemica. 2, 34-35.
Chester, N. and Marshak, D.R. (1993). Analytical Biochemistry. 209, 284-290.

Shipping Condition 
dry ice

User protocol

  25 µl Reaction 50 µl Reaction Final Concentration
I-5 2X High-Fidelity Master Mix 12.5 µl 25 µl 1X (see notes)
10 µM Primer A 1 µl 2 µl 400 µM
10 µM Primer B 1 µl 2 µl 400 µM
Template DNA as needed as needed see notes
50mM MgCl2 as needed as needed see notes
Water (ddH2O) up to 25 µl up to 50 µl  
Thermocycling Conditions      
3 Step PCR      
Step Temperature Time  
Initial Denaturation 98°C 2 minutes  
Denaturation 98°C 10 seconds 25-35 cycles
Annealing 55°C - 68°C 10-15 seconds  
Extension 72°C 10-15 seconds / kb  
Final Extension 72°C 1-5 minutes  
  4°C Hold  
2 Step PCR (see notes)      
Step Temperature Time  
Initial Denaturation 98°C 2 minutes  
Denaturation 98°C 10-15 seconds 25-35 cycles
Combined Annealing & Extension 72°C 15-30 seconds / kb  
Final Extension 72°C 1-5 minutes  
  4°C Hold  
Recommended DNA Template addition      
Genomic DNA 50-250 ng    
Plasmid DNA 1pg-10ng    
Viral DNA 1pg-10ng    
The final concentration of Mg2+ in 1X I-5 PCR Master Mix is 1.5 mM      
Add additional Mg2+ as needed in 0.5 mM increments      
Suggested final Mg2+ concentration ranges from 2 mM to 4 mM      
2 Step PCR      
Use of 2 Step PCR is recommended when the primer Tm values are >68°C      
PCR Product / Cloning      
The 2X I-5 PCR Master Mix results in PCR product with blunt ends      
Blunt-end cloning is recommended after PCR with this product      
For T/A-cloning, the PCR product should be purified before A-addition      


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