2× High-Fidelity PCR Master Mix (with Dye)
Based on 1 publication(s) in Google Scholar
MCE 2× High-Fidelity PCR Master Mix (with Dye) is a new type of ready-to-use high-fidelity PCR amplification master mix.During the reaction, only template and primers need to be added to quickly and efficiently complete the PCR process, reducing the risk of contamination during operation. The 1 mL is defined as the base specification. All larger sizes correspond to incremental volumes of this base.
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Storage :
-20℃, 2 years. Avoid repetitive freeze-thaw cycles.
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Description & Advantages
MCE 2× High-Fidelity PCR Master Mix (with Dye) is a new type of ready-to-use high-fidelity PCR amplification master mix. This product contains a modified new rapid high-fidelity DNA polymerase, dNTPs, and an optimized buffer system. The DNA polymerase exhibits 5’→3’ DNA polymerase activity and 3’→5’ exonuclease activity. During the reaction, only template and primers need to be added to quickly and efficiently complete the PCR process, reducing the risk of contamination during operation.
The two monoclonal antibodies added to this product can inhibit the polymerase activity and 3’→5’ exonuclease activity at room temperature. Additionally, an extension factor has been included to enable the DNA polymerase’s ability to amplify long fragments, allowing for the amplification of target fragment up to 13 kb, making it suitable for complex templates. The amplified products have blunt ends.
This product contains bromophenol blue dye, allowing for direct electrophoresis detection of the PCR products without the need to add DNA loading buffer.
Protocol
General Protocol
1. Preparation of the reaction system
Prepare the PCR reaction mixture on ice:
| Component | Volume | Final concentration |
|---|---|---|
| 2× High-Fidelity PCR Master Mix (with Dye) | 25 μL | 1× |
| PCR Forward Primer (10 μM) | 2.5 μL | 0.4 μM |
| PCR Reverse Primer (10 μM) | 2.5 μL | 0.4 μM |
| DNA | X μL | / |
| ddH2O | To 50 μL | / |
Recommended template amounts:
| Template type | Recommended amount for 1-10 kb target (50 μL reaction) |
|---|---|
| Genomic DNA | 100-400 ng |
| Plasmid / Viral DNA | 20 pg-40 ng |
| cDNA | 2-5 μL (no more than 1/10 of the total PCR volume) |
2. PCR program setup
Two-step PCR program (preferred)
| Step | Temperature | Time | Cycles |
|---|---|---|---|
| Initial denaturation | 98°C | 3 min | 1 |
| Denaturation | 98°C | 10 sec | 30-35 |
| Extension | 68°C | 30 sec/kb | 30-35 |
| Final extension | 72°C | 5 min | 1 |
Three-step PCR program (standard protocol)
| Step | Temperature | Time | Cycles |
|---|---|---|---|
| Initial denaturation | 98°C | 3 min | 1 |
| Denaturation | 98°C | 10 sec | 30-35 |
| Annealing | 60°C | 20 sec | 30-35 |
| Extension | 72°C | 30 sec/kb | 30-35 |
| Final extension | 72°C | 5 min | 1 |
Note: a. For standard PCR, initial denaturation is 98°C for 3 min; for high-GC templates it may be extended to 5-10 min;
b. Annealing temperature and time may be optimized according to primer Tm and actual amplification conditions; annealing times longer than 30 sec are not recommended;
c. Standard extension time is 30 sec/kb; for complex templates it may be extended to 60 sec/kb.
Gradient annealing program (for difficult-to-amplify genes)
| Step | Temperature | Time | Cycles |
|---|---|---|---|
| Initial denaturation | 98°C | 3 min | 1 |
| Denaturation | 98°C | 10 sec | 15 |
| Gradient annealing | 70°C→55°C (decrease 1°C per cycle) | 20 sec | 15 |
| Extension | 72°C | 30 sec/kb | 15 |
| Denaturation | 98°C | 10 sec | 20 |
| Annealing | 55°C | 20 sec | 20 |
| Extension | 72°C | 30 sec/kb | 20 |
| Final extension | 72°C | 5 min | 1 |
Bromophenol blue dye is included, so PCR products may be loaded directly onto an agarose gel.
Publications
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Journal Impact Factor
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Storage
-20℃, 2 years.
Avoid repetitive freeze-thaw cycles.
Components
| Components | HY-K0533A-1 mL | HY-K0533A-5 mL |
|---|---|---|
| 2× High-Fidelity PCR Master Mix (with Dye) | 1 mL | 1 mL × 5 |
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