Protocol for Taq 5X Master Mix PCR Reaction
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).
- 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
1 minute per kb
Final Extension 68°C 5 minutes Final Soak 4-10°C