Protocol for a Routine Taq PCR
Introduction
All components should be mixed and spun down prior to pipetting. These recommendations serve as a starting point; in order to maximize amplification the reaction conditions may require optimization (see Taq DNA Polymerase Guidelines for PCR Optimization protocol).
Protocol
- Prepare the following 50 µl reaction in a 0.5 ml PCR tube on ice:
* Due to the difficulties in pipetting small volumes of enzyme, Taq DNA Polymerase can be diluted in 1X reaction buffer. For example, 1 µl of Taq DNA Polymerase is mixed with 4 µl of 1X reaction buffer and 1 µl of that mixture is added to the reaction. Enzyme diluted 1X reaction buffer should not be stored for future use.
Component 25 μl reaction 50 μl reaction Final Concentration 10X ThermoPol or Standard Taq Reaction Buffer 2.5 µl 5 μl 1X 10 mM dNTPs 0.5 µl 1 μl 200 µM 10 µM Forward Primer 0.5 µl 1 μl 0.2 µM (0.05–1 µM) 10 µM Reverse Primer 0.5 µl 1 μl 0.2 µM (0.05–1 µM) Template DNA variable variable <1,000 ng Taq DNA Polymerase* 0.125 µl 0.25 µl 1.25 units/50 µl PCR 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:
CYCLE STEP
TEMP
TIME
CYCLES Initial Denaturation
95°C
30 seconds
1
Denaturation
Annealing
Extension
95°C
45-68°C
68°C
15-30 seconds
15-60 seconds
1 minute per kb
30
Final Extension 72°C
5 minutes
1 Hold 4°C
∞
