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I-5™ Hi-Fi HotStart DNA Polymerase
I-5™ Hi-Fi HotStart DNA Polymerase

I-5 Hi-Fi DNA HotStart Polymerase is a high-fidelity and fast DNA polymerase.

 
Name Cat# Description Price Qty
I-5™ Hotstart DNA Polymerase
I5HD-100
250 Units, 2.5 U/μl
$147.50
I-5™ Hotstart DNA Polymerase
I5HD-200
1,250 Units, 2.5 U/μl
$572.50
I-5™ Hotstart DNA Polymerase
I5HD-OEM
any size
please inquire

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 10kb 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.

Features

  • Fast – 1 kb / 10-15 seconds
  • High fidelity  – 1 error per 110,538 nt
  • Robust – high inhibitor tolerance
  • High yields – high efficiency
  • Long range PCR – ~ 10kb human genomic DNA

Source
E. coli

Applications

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

Supplied with
5X Reaction Buffer (1ml, 5X MgClincluded )

50mM MgCl2 (1ml) (for optimization)

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.

Experimental Data

High Fidelity DNA Polymerase Comparison of I5 (MCLAB), KAPA (Roche), and Q5 (NEB)

DNA polymerase fidelity is estimated by the error rate, i.e., the percentage of base substitutions per PCR cycle (given a replication efficiency of 2.0). The error rates of DNA Polymerase I5 (MCLAB), KAPA (Roche) and Q5 (NEB) are measured by both Sanger sequencing and Next Generation Sequencing (Illumina_MiSeq) (Table 1).

DNA Polymerase

I5

KAPA

Q5

Sanger Sequencing

(error /100,000bp)

0.9

1.7

0.9

NGS_Illumina

(error /100,000bp)

1.6

1.3

2.5


Conclusion:

The fidelity of MCLAB’s I5 DNA polymerase is ultra-high, with an average of 1.25 errors per 100,000bp from both Sanger Sequencing and NGS_Illumina, which is at least equivalent to that of KAPA or Q5.

Methods:

In this assay, I5 was examined to determine its fidelity compared to KAPA  and Q5 DNA polymerase. A 2000 bp template was PCR amplified with either I5, or KAPA, or Q5 DNA Polymerase for 35 cycles. The PCR error rates were determined by Sanger sequencing (Figure 1) and NGS_Illumina (Figure 2). The raw data was normalized by the following equation:

e = (f⋅2) / n

where f is the percentage of errors measured after PCR, and n is the number of actual PCR cycles.  

I5-001.png

Figure 1. PCR polymerase fidelity estimation by Sanger Sequencing.

I5-002.png

Figure 2. PCR polymerase fidelity estimation by Next Generation Sequencing (Illumina_MiSeq).

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 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      

 

Manual & Protocols



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