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Taq Pol (+)MgCl2 | Taq DNA Polimerase - Cellco
Taq Pol (+)MgCl2
Ficha técnica
| Código | Taq Pol (+)MgCl2 |
| Categoria | PCR Convencional |
| Marca | Cellco |
Descrição Geral
Taq Pol (+)MgCl2 | Taq DNA Polimerase - Cellco
For in vitro use only!
Definição de unidade: One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nmoles of dNTPs into an acidinsoluble form in 30 minutes at 70 °C.
Envio: Shipped on blue ice
Condições de armazenamento: Store at -20 °C
Avoid freeze/thaw cycles
Validade: 12 months
Forma: Liquid
Concentração: 5 units/μL
Descrição:
Taq Pol is recommended for use in routine PCR reactions. It is optimized for high specificity and guarantees minimal byproduct formation. The buffer system is recommended for plate based PCR and automated pipetting. The enzyme replicates DNA at 72 °C. It catalyzes the polymerization of nucleotides into duplex DNA in 5' → 3' direction in the presence of magnesium. It also possesses a 5' → 3' polymerization-dependent exonuclease replacement activity but lacks a 3' → 5' exonuclease activity.
PCR Reaction Setup:
The PCR procedure below shows appropriate volumes for a single 50 μL reaction. For multiple reactions, prepare a master mix of components common to all and then dispense appropriate volumes into each PCR reaction tube prior to adding template DNA and primers. Thaw, mix, and briefly centrifuge each component before use.
Add the following components to a microcentrifuge tube:
1. Prepare PCR master mix
Note: Consider the volumes for all components listed next steps to determine the correct amount of water required to reach your final reaction volume.
| Component | 50 μl assay | Final conc. |
| PCR grade water | to 50 μl | - |
| 10x Reaction Buffer Complete | 5 μl | 1x |
| dNTP Mix 10 mM | 1 μl | 200 μM |
| Taq DNA Polymerase (5 U/μl) | 0,25-0,5 μl | 1,25-2,5 U/assay |
| template DNA | x μl | <500 ng |
Mix and briefly centrifuge the components.
2. Add template DNA and primers
| Component | 50 μl assay | final conc. |
| Forward primer (10 μM) | 0,5 – 2,5 μl | 0,1 – 0,5 μM |
| Reverse primer (10 μM) | 0,5 – 2,5 μl | 0,1 – 0,5 μM |
| Template DNA | x μl | 10 pg – 1 μg* |
*genomic DNA: 1 ng – 1 μg; plasmidial DNA: 1 pg – 1 ng
Cap each tube, mix, and briefly centrifuge the content.
3. Optimization of MgCl2 concentration:
The 10x reaction buffer contain 20 mM MgCl2, a recommended concentration for most applications. For an individual optimization add MgCl2 stock solution as shown in the table below.
| MgCl2 Final Concentration | 2,5 mM | 3 mM | 4 mM |
| MgCl2 Stock volume to 50 μl | 1 μl | 2 μl | 4 μl |
4. Incubate reactions in a thermal cycler
Recommended cycling conditions:
| Initial denaturation | 95 °C | 1 – 3 min | 1x |
| Denaturation | 95 °C | 15 - 30 sec | 30 cycles |
| Annealing** | 45 - 68 °C | 15 - 30 sec | 30 cycles |
| Elongation*** | 72 °C | 1 min/kb | 30 cycles |
| Final extension (optional) | 72 °C | 1 – 2 min/kb | 1x |
| Hold | 4 – 8 °C | ∞ | 1x |
**The annealing temperature depends on the melting temperature of the primers used
***The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.
For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.
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