I-5™ Hi-Fi HotStart DNA Polymerase
$0.00 – $648.65
I-5 Hi-Fi DNA HotStart Polymerase is a high-fidelity and fast DNA polymerase.
| SKU | OPTIONS | Price | Quantity | ||
|---|---|---|---|---|---|
| I5HD-100 | 250 Units, 2.5 U/ul | $167.12 | |||
| I5HD-200 | 1,250 Units, 2.5 U/ul | $648.65 | |||
| I5HD-OEM | any size | Please inquire |
- Description
- Additional information
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Description
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:
expressed in E. coli
Applications:
- Hot Start PCR
- Routine PCR
- Fast PCR
- High throughput PCR
- Genotyping
Supplied with:
5X Reaction Buffer (1ml, 5X MgCl2 included )
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.
Figure 1. PCR polymerase fidelity estimation by Sanger Sequencing.
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 | |||
Additional information
| OPTIONS | 250 Units, 2.5 U/ul, 1,250 Units, 2.5 U/ul, any size |
|---|
Manual & Protocols
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