TaqSure 2X Master Mix
Product Components
| Components | Component Number | Size-1 | Size-2 |
|---|---|---|---|
| TaqSure 2X Master Mix | MZ20408 | 1 mL | 1 mL × 5 |
Product Description
TaqSure DNA polymerase is an ideal enzyme for high-fidelity PCR with excellent processivity. It is a novel engineered enzyme with a unique structure. TaqSure DNA polymerase contains a recombinant synthetic enhanced domain to increase fidelity and extension speed. The antibody-mediated hot-start feature significantly inhibits non-specific amplifications at room temperature. TaqSure is one of the thermostable DNA polymerases with strong 3’-5’ exonuclease activity (proofreading activity), which results in extremely high fidelity—approximately 100× that of conventional Taq. The TaqSure 2X Master Mix is an ideal product with good amplification efficiency for diverse templates including animals, plants, cDNA, etc. TaqSure 2X Master Mix contains TaqSure DNA polymerase, dNTPs, and an optimized buffer system. It only requires the addition of primers and templates to perform amplification, thereby reducing pipetting operations, significantly controlling cross-contamination among samples, and improving detection throughput and reproducibility of results. The amplification system contains protective agents that keep TaqSure 2X Master Mix stable in activity after repeated freezing and thawing.
Storage
Store at −20 °C
5´-3 ´exonuclease acvity
No
3´-5 ´exonuclease acvity
Yes
Product End
Blunt end
Operaon Descripon
Standard Protocol
It is recommended to prepare all reaction components on ice, and then quickly transfer the reaction system to a thermocycler preheated to 98°C.
All components should be mixed and collected at the bottom of a tube with a quick spin before use. Add TaqSure 2X Master Mix at the end to prevent primer degradation by its strong 3´-5´ exonuclease activity.
Note: The TaqSure DNA polymerase requires special reaction conditions different from other polymerase protocols. Please refer to the recommended reaction conditions below for better amplification yields.
Recommended Reaction:
| Component | 25 μL Reaction | 50 μL Reaction | Final Concentration |
|---|---|---|---|
| TaqSure 2X Master Mix | 12.5 µL | 25 µL | 1X |
| Forward Primer (10 μM) | 0.5 µL | 1 µL | 0.2 µM |
| Reverse Primer (10 μM) | 0.5 µL | 1 µL | 0.2 µM |
| DNA Template* | Variable | Variable | <300 ng |
| Nuclease-free Water | to 25 µL | to 50 µL | N/A |
*Note: The optimal reaction concentration varies with different DNA templates. Please refer to the basic principles of PCR below.
Recommended PCR Program:
| Step | Temp | Time | Cycles |
|---|---|---|---|
| Initial Denaturation | 98°C | 3 min | 1 |
| Denaturation | 98°C | 10 s | 25–35 |
| Annealing | 55–65°C | 20–30 s | 25–35 |
| Extension | 72°C | 30–60 s/kb* | 25–35 |
| Final Extension | 72°C | 1–5 min | 1 |
| Hold | 4–12°C | – | 1 |
*Note: Properly extending the extension time can improve the amplification yield. For complex amplification templates, such as genomic DNA, it is recommended to extend at a speed of 60 s/kb. For more recommended conditions, please refer to the basic principles of PCR below.
PCR Principles
1. Template
High-quality purified DNA templates are important for high-fidelity PCR reactions. The recommended DNA template amounts with different complexity are listed below (for a 50 µL reaction):
| DNA Type | Input Amount |
|---|---|
| Plants, animals, and human gDNA | 10 ng–100 ng |
| E. coli, lambda gDNA | 500 pg–200 ng |
| Plasmid DNA | 1 pg–10 ng |
Note: If the DNA template is obtained from a cDNA synthesis reaction, the template volume should be less than 10% of the total reaction volume. For long fragment amplification, the amount of template input should be increased appropriately.
2. Primers
Oligonucleotide primers are typically 20–40 nucleotides in length with a GC content of 40–60%. Primers can be designed and analyzed using software such as Primer3. The final concentration of each primer in the PCR reaction system should be in the range of 0.1–1 μM.
3. Denaturation
The DNA template can be fully denatured when the initial denaturation time is set to 3 min. Generally, the recommended denaturation condition for low-complexity DNA templates is 98°C for 5–10 s.
Here’s your Annealing, Extension, Cycles, PCR Products, and Complex Templates guidelines formatted into a clear, readable paragraph style while keeping the exact same content:
4. Annealing
The annealing temperature of TaqSure DNA polymerase is usually higher than other PCR polymerases. Generally, primers longer than 20 nt are annealed at (lower primer Tm + 3)℃ for 10–30 s; when the primers are shorter than 20 nt, an annealing temperature equivalent to the lower primer Tm is used. When using a new primer set for PCR reaction, we recommend a gradient PCR to determine the optimal annealing temperature. In a two-step amplification protocol, the annealing temperature should be set to the extension temperature.
5. Extension
The recommended extension temperature is 72℃. The extension time depends on the length and complexity of the amplicon. For low-complexity amplicons (plasmid DNA), the extension condition is 30 s/kb. For high-complexity amplicons, it is recommended to increase the extension time to 60 s/kb. In some cases, the extension time for cDNA templates should be less than 1 min/kb.
6. Cycles
To obtain sufficient yield of PCR products, 25–35 cycles are recommended.
7. PCR Products
TaqSure DNA polymerase produces blunt-end PCR products, which might be directly used in sequential blunt-end cloning.
8. Complex Templates
For complex templates that cannot be amplified by conventional PCR (such as long fragments, uneven Tm distribution, templates with special structures), you can try the two-step method or the touchdown method.
Recommended Two-Step PCR Program
| Step | Temp | Time | Cycles |
|---|---|---|---|
| Initial Denaturation | 98°C | 3 min | 1 |
| Denaturation | 98°C | 10 s | 25–35 |
| Annealing/Extension* | 65–72°C | 60 s/kb | 25–35 |
| Final Extension | 65–72°C | 1–5 min | 1 |
| Hold | 4–12°C | – | 1 |
*Note: In general, 68°C is recommended, but it can be changed according to the Tm value.
Recommended Touchdown PCR Program:
| Step | Temp | Time | Cycles |
|---|---|---|---|
| Initial Denaturation | 98°C | 3 min | 1 |
| Denaturation | 98°C | 10 s | 5 |
| Annealing | 74°C | 60 s/kb | 5 |
| Denaturation | 98°C | 10 s | 5 |
| Annealing | 72°C | 60 s/kb | 5 |
| Denaturation | 98°C | 10 s | 5 |
| Annealing | 70°C | 60 s/kb | 5 |
| Denaturation | 98°C | 10 s | 25 |
| Annealing | 68°C | 60 s/kb | 25 |
| Final Extension | 72°C | 1–5 min | 1 |
| Hold | 4–12°C | – | 1 |