2x HiFi PCR Master Mix

• 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

2x HiFi PCR Master Mix is a high efficiency, high-fidelity and low bias PCR master mix for NGS library amplification.

2x HiFi PCR Master Mix is supplied with HiFi Enhancer (cat. no. M0313L).

Mix properties

• Storage temperature: –25°C to –15°C

The 2x HiFi PCR Master Mix (P7670L) 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 a limited amount of starting material usually require PCR amplification to generate sufficient templates for library quantification and sequencing. Libraries amplified using the 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.

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.

* 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.

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.

Post-capture PCR enrichment

The 2x HiFi PCR Master Mix is compatible with target enrichment workflows such as those utilizing bead-based hybridization capture probes and panels. The following instructions are validated for PCR amplification of targets enriched using xGen Lockdown Probes and reagents from IDT. In this workflow, the targets to be amplified are immobilized on Dynabeads M-270 Streptavidin beads. If other types of capture beads are used, the reaction may need to be optimized accordingly. 

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	45 sec	1
   Denaturation	98°C	15 sec	As required*
   Annealing	60°C	30 sec
   Extension	72°C	30 sec
   Final Extension	72°C	1 min	1
   Hold	4°C	∞	N/A

   * The number of PCR cycles should be optimized according to capture probe supplier’s guideline
2. Prepare the post-capture amplification reaction mix in a separate tube per the table below.

   Components	Volume for 1 reaction (µL)
   Beads with Captured DNA Template	X
   2x HiFi PCR Master Mix	25
   Primer Mix*	Y
   HiFi Enhancer†	2
   Nuclease-free Water	23-X-Y
   Total Volume	50

   * A final concentration of 0.5 μM each primer for on-bead amplification. Please follow manufacturer/supplier’s instructions. 
   † 2-2.5 μL of the HiFi Enhancer should be added to each reaction to ensure high efficiency on-bead amplification. When using a higher volume of beads, scale the amount of HiFi Enhancer accordingly. 
3. Pipette up and down to ensure mixing of components. Spin down gently but ensure the beads remain in solution.
4. Transfer the reaction tube to the pre-heated thermal cycler (98°C). Resume the cycling program.
5. When the thermal cycler program is complete and sample block has returned to 4°C, remove the sample from block and proceed to immediately proceed to Post-Amplification Cleanup using QIAseq beads or other desired purification method. 
6. Validate and quantify the library using gel electrophoresis, qPCR and/or Bioanalyzer.

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.

This product is available for molecular biology applications such as:

• NGS library amplification