I-5™ Hi-Fi DNA Polymerase

Size :

250 Units, 2.5 U/μl

More info

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.

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