I-5™ Hi-Fi HotStart DNA Polymerase

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General Description
I-5 Hi-Fi DNA HotStart Polymerase is an ultra-fast and high-fidelity DNA polymerase. It provides robust amplification of difficult templates including plasmids, BACS, genomic DNA, and lambda DNA. It allows the amplification of up to 8kb human genomic DNA and up to 21kb lambda DNA. It has an extension speed of 1 kb / 10-15 seconds depending on template type. This allows users to save time by speeding up PCR reactions and provides higher fidelity than Taq or Pfu. The enzyme contains a HotStart mechanism that inactivates the enzyme until it is heated. This allows users to setup PCR reactions at room temperature without worrying about primer dimers or non-specific preamplification.

Feature
• Fast – 4X faster than hot start Taq
• Robust – high inhibitor tolerance
• High yields – high efficiency
• Long PCR products

Source
E. coli

Applications
Hot Start PCR
Routine PCR
Fast PCR
High throughput PCR
Genotyping

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 8.0 @ 25 C

Storage Condition
-20ºC

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.

Reference
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

Instructions

	25 µl Reaction	50 µl Reaction	Final Concentration
I-5 5X Buffer	5 ul	10 ul	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
I-5 Enzyme	0.5 - 1 ul	1 - 2 ul
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 2 Step PCR is recommended when the primer T_m 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