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Protocol for Taq 5X Master Mix PCR Reaction |  | | Protocol for Taq 5X Master Mix PCR Reaction |
Overview
These recommendations serve as a starting point; in order to maximize amplification the reaction conditions may require optimization (see "Taq 5X Master Mix Guidelines for PCR Optimization" protocol).
Protocol
- Thaw Taq 5X Master Mix at room temperature and mix well by inverting several times before use.
- Prepare the following reaction in a thin-walled PCR tube on ice:
| Component | 25 µl reaction | 50 µl reaction | Final Conc. | | Taq master Mix (5X) | 5 µl | 10 µl | 1X | | Upstream Primer (10 μM stock) | 0.5 µl | 1 µl | 0.2 µM | | Downstream Primer (10 μM stock) | 0.5 µl | 1 µl | 0.2 µM | | Template DNA | determined bu user | determined by user | <500 ng | | Nuclease-free water | to 25 µl | to 50 µl | | - Gently mix the reaction and spin down in microcentrifuge.
If the thermocycler does not have a heated cover, add one drop of mineral oil to the reaction tube to prevent evaporation. - Cycling conditions for a routine PCR reaction:
| Initial Denaturation | 94-95°C | 1 minute | | 25-40 Cycles | 94-95°C
45-70°C
68°C | 20 seconds
30 seconds
1 minute per kb | | Final Extension | 68°C | | 5 minutes | | Final Soak | 4-10°C | | |
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