Description
Fast Taq PCR master mix
GenieTaq Rapid 2X Master Mix combines Taq DNA polymerase with an extension enhancer for fast amplification at 15 sec/kb, and as fast as 1 sec/kb for fragments within 1 kb. It is ideal for fast screening and colony PCR. Just add primers and template.
The mix contains Taq DNA polymerase, an elongation-promoting factor, dNTPs and an optimised buffer. It amplifies fragments of up to 5 kb from genomic DNA and up to 10 kb from plasmid or λDNA. A protective agent keeps the mix stable through repeated freeze-thaw cycles, and the premixed format cuts pipetting for better throughput and reproducibility. Products carry a 3'-dA overhang and can be cloned directly into T vectors, or used with seamless and TOPO cloning kits. For research use only.
Key Features
| Rapid amplification | Extension at 15 sec/kb, and as fast as 1 sec/kb for fragments ≤1 kb. | |
| Good stability | Performance remains stable after 50 freeze-thaw cycles. | |
| Ready-to-use | The 2X master mix format simplifies the workflow and minimises pipetting. | |
| TA cloning ready | Products carry a 3'-dA overhang for direct cloning into T vectors. |
Specifications
| Format | 2X ready-to-use master mix (Taq DNA polymerase, elongation-promoting factor, dNTPs, buffer) |
| Extension speed | 15 sec/kb; up to 1 sec/kb for fragments ≤1 kb |
| Amplicon length | Up to 5 kb (genomic DNA); up to 10 kb (plasmid, λDNA) |
| Application | Fast PCR, screening and colony PCR |
| Product ends | 3'-dA overhang |
| Shelf life | 2 years |
Components
| Components | Amount |
| GenieTaq Rapid 2X Master Mix | 5 × 1 ml / 15 × 1 ml / 50 × 1 ml / 10 × 5 ml |
Storage
Store at -30 ~ -15°C.
- Prepare the reaction system
- Set up a 50 μl reaction as follows:
| Components | Volume |
| ddH2O | To 50.0 μl |
| GenieTaq Rapid 2X Master Mix | 25.0 μl |
| Primer 1 (10 μM) | 2.0 μl |
| Primer 2 (10 μM) | 2.0 μl |
| Template DNA* | x μl |
*Recommended template amount in a 50 μl reaction:
| Template type | Amount |
| Animal and plant genomic DNA | 0.1–1 μg |
| E. coli genomic DNA | 10–100 ng |
| cDNA | 1–5 μl (≤1/10 of the total PCR volume) |
| Plasmid DNA | 0.1–10 ng |
| λDNA | 0.5–10 ng |
- Run the reaction program
| Step | Temperature | Time | Cycles |
| Initial denaturationa | 95°C | 3 min | 1 |
| Denaturation | 95°C | 15 sec | 30–35 |
| Annealingb | 60°C | 15 sec | 30–35 |
| Extensionc | 72°C | 15 sec/kb | 30–35 |
| Final extension | 72°C | 5 min | 1 |
a These conditions suit most reactions. For templates with complex structure, extend initial denaturation to 5–10 min.
b Set the annealing temperature 3–5°C below the primer Tm. For complex templates, adjust the annealing temperature and extend the extension time.
c For higher yields, use 2–5 sec for products shorter than 1 kb, or extend to 20–30 sec/kb for products longer than 1 kb.
Q1: What should I do if there is no amplification or the yield is low?
- Optimise primer design; run an annealing temperature gradient to find the optimum; increase primer concentration; increase the extension time; increase cycles to 35–40; and use high-purity template. Use less crude sample; for other samples, follow the recommended amount and increase it moderately.
Q2: How can I reduce nonspecific products or smeared bands?
- Optimise primer design; raise the annealing temperature in 2°C steps up to 65°C; decrease the final primer concentration to 0.2 μM; shorten the extension time if nonspecific bands are larger than the target; reduce cycles to 25–30; use high-purity template; and adjust the template amount to the recommended range.
Q3: How fast can I run the extension step?
- The standard extension time is 15 sec/kb, and fragments within 1 kb can be amplified at up to 1 sec/kb. For higher yields, use 2–5 sec for products shorter than 1 kb or 20–30 sec/kb for products longer than 1 kb.
