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Address: Room 305, Building A, No. 179, Guangpu East Road, Huangpu District, Guangzhou, Guangdong
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Guangzhou Dongsheng Biotech Co., Ltd |
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NGS Multiplex PCR Master Mix II, 2X
The NGS Multiplex PCR Master Mix II contains all of the components for NGS multiplex PCR (except for primers and templates) in a single tube, including chemically modified ultra-high fidelity DNA Polymerase, which has 100X amplification fidelity of Taq DNA Polymerase. The PCR products have an ultra-low error rates, and is very suitable for the detection of tumor ctDNA and MRD.
| Cat. No. | Contents | Storage Conditions |
| NM2001 | NGS Multiplex PCR Master Mix II, 2X, 40 reactions |
Store unopened at –15°C to –25°C until the expiration date on the label. After opening, the master mix may be stored at –15°C to –25°C until the expiration date on the label, or at 4°C for up to 30 days. The GC Enhancer must be kept at –15°C to –25°C. |
| • NGS Multiplex PCR Master Mix II, 2X (1 × 1 mL) | ||
| • GC Enhancer (1 × 0.25 mL) | ||
| NM2002 | NGS Multiplex PCR Master Mix II, 2X, 400 reactions | |
| • NGS Multiplex PCR Master Mix II, 2X (10 × 1 mL) | ||
| • GC Enhancer (2 × 1 mL) | ||
| NM2003 | NGS Multiplex PCR Master Mix II, 2X, 2000 reactions | |
| • NGS Multiplex PCR Master Mix II, 2X (5 × 10 mL) | ||
| • GC Enhancer (1 × 10 mL) |
Protocol
Note: Before setting up the PCR reactions, prepare a Primer Mix II with 0.5 µM of each primer.
1. Allow all reagents to thaw completely. Mix gently by inverting the tube. Spin briefly. Put all reagents on ice.
2. Using a 96-well optical reaction plate, add the following to one well per sample:
| Component | Volume or Mass | Final Concentration |
| NGS Multiplex PCR Master Mix II, 2X | 25 µL | 1X |
| Primer Mix II(0.5 µM each) | 5 µL | 50 nM each primer[1] |
| Template DNA | 0.1–0.2 µg | 2–4 ng/µL |
| GC Enhancer | 0 or 6 µL [2] | 0 or 12% |
| Nuclease-free water | Adjust to 50 µL | n/a |
[1] The final concentration of each primer in a typical PCR is between 0.05-0.4 μM. In most cases, a final concentration of 0.15 μM gives satisfactory results. Increasing the primer concentration up to 0.4 μM may increase the yield.
[2] Use GC Enhancer only when high GC content targets cannot be amplified under standard conditions.
3. Seal the reaction plate with Clear Adhesive Film.
Amplify DNA for Analysis
Choose an amplification protocol based on your analysis method
Amplify for analysis by agarose gel electrophoresis
1. Configure the run method as outlined in your instrument’s user manual. Use the following parameters:
Three-step reaction system:
| Step | Time | Temperature (°C) |
| Hold | 1 min | 98 |
| 25-35 Cycles | 15 sec | 98 |
| 30 sec | 58 | |
| 30 sec | 72 | |
| Hold | 2 min | 72 |
| Hold | ¥ | 4 |
Two-step reaction system(Tm>60℃):
| Step | Time | Temperature (°C) |
| Hold | 1 min | 98 |
| 25-35 Cycles | 15 sec | 98 |
| 60 sec | 60 | |
| Hold | 2 min | 72 |
| Hold | ¥ | 4 |
2. Mix well and briefly spin the reaction plate.
3. Load the reaction plate into the instrument, and start the run. See your instrument’s user manual for detailed instructions on how to load and run the plate.
4. Analyze the data according to the instructions in the user manual for your gel electrophoresis instrument.
Amplify for CfDNA Sequencing
| Component | Volume |
| NGS Multiplex PCR Master Mix II, 2X | 12.5 µL |
| Primer Mix II | 2 µL |
| cfDNA | X µL |
| Nuclease-free water | To 25 µL |
1. Configure the run method as outlined in your instrument’s user manual. Use the following parameters:
Three-step reaction system:
| Step | Time | Temperature (°C) |
| Hold | 1 min | 98 |
| 25-35 Cycles | 15 sec | 98 |
| 30 sec | 58 | |
| 30 sec | 72 | |
| Hold | 2 min | 72 |
| Hold | ¥ | 4 |
Two-step reaction system(Tm>60℃):
| Step | Time | Temperature (°C) |
| Hold | 1 min | 98 |
| 25-35 Cycles | 15 sec | 98 |
| 60 sec | 60 | |
| Hold | 2 min | 72 |
| Hold | ¥ | 4 |
2. Mix well and briefly spin the reaction plate.
3. Load the reaction plate into the instrument, and start the run. See your instrument’s user manual for detailed instructions on how to load and run the plate.
GDSBio is a high-tech company focused on the development, production and marketing of quality life science products. The company has a complete product line, with PCR technology as the core, focusing on ordinary PCR, fluorescent quantitative PCR, NGS library construction, nucleic acid electrophoresis and other molecular biology technologies, and has developed molecular scientific research reagents, molecular in vitro diagnostic raw materials, nucleic acid extraction and detection reagents and other products.
