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Read moreGeneral 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.
Feature
• 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
Source
E. coli
Applications
PCR
Cloning
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
-20°C
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.
References
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
Instructions | |||
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 | ||
Notes | |||
Recommended DNA Template addition | |||
Genomic DNA | 50-250 ng | ||
Plasmid DNA | 1pg-10ng | ||
Viral DNA | 1pg-10ng | ||
Mg2+ | |||
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 |