The CD HiFi Amplification Mix is a high-fidelity PCR amplification master mix that can be used to amplify NGS libraries. The master mix is based on HiFi DNA Polymerase, which was manufactured from Pfu DNA Polymerase and has a high yield and fidelity. With extremely high amplification efficiency, low amplification mismatch rate, and extensive adaptability of the template, this enzyme's sensitivity has improved significantly. It significantly enhances amplification output and ensures that library amplification is genuine.
Application:
HiFi Amplification Mix is applicable to High-fidelity PCR amplification, such as high-throughput sequencing library PCR amplification.
Storage:
The Kit should be stored at -30~-15°C.
Components:
Specifications:
| Application | HiFi Amplification Mix is applicable to High-fidelity PCR amplification, such as high-throughput sequencing library PCR amplification. |
- Thaw PCR Primer Mix 3 and HiFi Amplification Mix, and mix thoroughly. Prepare the reaction solution in a sterile PCR tube as follows:
- Mix thoroughly by gently pipetting up and down. DO NOT Vortex! Spin down briefly.
- Put the tube in a PCR instrument and run the PCR program according to the handbook.
- For size selection, please refer to CD Universal DNA Library Prep Kit for Illumina (DP001).
| Reagents | Volume of Sample |
| Purified or Size-Selected Adapter-Ligated Library | X µl |
| PCR Primer Mix 3 for Illumina* | 5 µl |
| HiFi Amplification Mix | 25 µl |
| Total Volume | To 50 µl |
• The PCR Primer Mix 3 is suitable for the amplification of all Illumina libraries flanked by the P5 and P7 flow cell sequences. User-supplied primer mixes may be used in combination with incomplete or custom adapters. Each primer should be used at a final concentration of 5μM-20μM each.
• If CD Multiplex Oligos S1/S2 for Illumina (DDI001/DDI002) is used, the primer mix should be 2.5 μl of i5 PCR Primer DM5XX and 2.5 μl of i7 PCR Primer DM7XX.
• *For non-Illumina sequencing platforms, the corresponding library amplification primers must be changed.
Notes:
a. This process is to amplify the purified or size-selected adapter ligation products. Whether to proceed with this step depends on the amount of input DNA, whether adapters are in complete length, and the need of application. If adapters are not in complete length (i.e. DDI001/ DDI002), this step is necessary. If adapters are in complete length (i.e. DSI001/ DSI002), for input DNA < 50 ng, amplification is recommended. Skip this step if input DNA is ≥ 50 ng or there is no need for library amplification.
b. All operations should be on the ice, invert HiFi Amplification Mix up and down after thawing, immediately put it back to -20℃ after use.
c. It is recommended to use a purified high quality library template and follow the instructions.
d. For special libraries, such as low-quality libraries and long-fragment libraries, the annealing temperature, extension time, and amplification cycle numbers can be adjusted according to the reaction procedure to obtain the best amplification results.
e. When adjusting the extension time, it should not exceed 1 min/kb.
