Description
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. 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. 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. 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 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. Pipette up and down to ensure mixing of components. Spin down gently but ensure the beads remain in solution. Transfer the reaction tube to the pre-heated thermal cycler (98°C). Resume the cycling program. 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. 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.
