I-5™ Hi-Fi HotStart DNA Polymerase

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

SKU OPTIONS 价格
I5HD-100 250 Units, 2.5 U/ul ¥1,755.00
I5HD-200 1,250 Units, 2.5 U/ul ¥6,811.00
I5HD-OEM any size Please inquire

描述

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.

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

其他信息

OPTIONS

250 Units, 2.5 U/ul, 1,250 Units, 2.5 U/ul, any size

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