HiFi PCR Master Mix

OEM by QIAGEN offers bulk manufacturing of HiFi PCR Master Mix in custom formulations.

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HiFi PCR Master Mix

Cat. No. / ID:  P7670-FIN

For one reaction (evaluation pack): 2x HiFi PCR Master Mix (25 µl).
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The HiFi PCR Master Mix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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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.

Related NGS DNA library preparation products
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)
Certificates of Analysis (1)