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Features
- High sensitivity and improved amplification efficiency in NGS applications
- Exhibits minimal bias in DNA library amplification
- Excellent genome coverage even in extremely high GC regions
Product Details
HiFi PCR Master Mix is a high efficiency, high-fidelity and low bias PCR master mix for NGS library amplification.
SDS is available on request.
Performance
Mix properties
- Storage temperature: –25°C to –15°C
Principle
The 2X HiFi PCR Master Mix (P7670) is a ready-to-use solution that contains all the components for NGS library amplifications, including a hot-start HiFi DNA polymerase in optimized buffer to ensure high efficiency, high fidelity and low bias amplifications. NGS libraries made with limited amount of starting materials usually require PCR amplification to generate sufficient templates for library quantification and sequencing. Libraries amplified using he 2X HiFi PCR Master Mix exhibit minimal bias, low error rate and excellent genome coverage even in extremely low or high GC regions. These features help to minimize sequencing bias, reduce duplication rate, and improve data quality and coverage uniformity.
Procedure
1. Program a thermal cycler with the parameters listed in the table below. Set the instrument’s heated lid to 105°C. When the thermal cycler block reaches 98°C, pause the program.
| Step | Temperature | Incubation time | Cycle number |
| Initial denaturation | 98°C | 2 minutes | 1 |
| Denaturation | 98°C | 20 seconds | As required† |
| Annealing | 60°C * | 30 seconds | |
| Extension | 72°C | 30 seconds‡ | |
| Final extension | 72°C | 1 minute | 1 |
| Holding | 4°C | Hold | 1 |
* Annealing temperature optimization may be necessary.
† Amplification cycle number needs to be adjusted based on DNA template concentration, primer concentrations and a DNA library yield sufficient for downstream applications.
‡ 30–60 sec/kb is recommended when deciding extension time.
2. Prepare the PCR in a new tube on ice by combining the DNA template, 2X HiFi PCR Master Mix and customer-supplied primers as described in the table below. Mix well by pipetting up and down 8–10 times. Volumes can be scaled as needed.
| Components | Volume for one reaction (µl) |
| DNA template | X |
| 2X HiFi PCR Master Mix | 25 |
| Primer mix | Y |
| Nuclease-free water | 25–X–Y |
| Total | 50 |
3. Pulse-spin the sample tube and immediately transfer to the pre-heated thermal cycler (98°C). Resume the cycling program.
4. When the thermal cycler program is complete and sample block has returned to 4°C, remove the sample from block and immediately proceed to post-amplification cleanup using AMPureXP beads or other desired purification method.
5. Validate and quantify the library using gel electrophoresis, qPCR and/or Bioanalyzer.
| Cat. no. | Description |
| Y9410L | 5X WGS Fragmentation Mix |
| Y9420L | 5X ER/A-Tailing Enzyme Mix |
| L6030-W-L | T4 DNA Ligase (NGS) |
Quality control analysis
HiFi PCR Master Mix is tested functionally by amplification of a DNA library prepared from mixed bacterial genomic DNA with GC-content of 10–80%. The differences in library yield and profile among different lots must not exceed 15%. Sequencing of the amplified library must yield mapped reads >90% and normalized coverage between 0.7 and 1.3 across the full GC spectrum.
Applications
This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.
- NGS library amplification
Resources
Safety Data Sheets (1)