How do I optimize a Polymerase Chain Reaction?

There are several things one can do to optimize a PCR reaction for GC-rich templates. One way to optimize is to increase the denaturation temperature. Use higher temperatures (like 98°C instead of 94°C or 95°C) to ensure the complete separation of DNA strands. Another thing to do is to choose primers with higher melting temperatures. Select primers with a melting temperature greater than 68°C. This ensures they bind securely to the DNA template even at higher annealing temperatures. One may also shorten annealing times as briefly as possible.

For AT-rich templates, it is recommended to use the same polymerase (e.g. EmeraldAmp GT PCR Master Mix,) as for GC-rich templates as they are also suitable to them. Additionally, for templates with AT content exceeding 80-85%, the extension temperature can be decreased from the standard 72°C to a range of 65-60°C. Studies suggest that DNA replication remains reliable at these reduced temperatures.