5 Tips for Setting Up Your PCR

Choose a polymerase that matches your experimental requirements. Consult the DNA polymerase selection chart at confidentialpcr.com if you need guidance on which polymerase best suits your specific needs.

Design primers carefully to reduce troubleshooting time, ensuring they are 20-30 nucleotides in length with 40-60% GC content. Verify that both forward and reverse primers have melting temperatures within 5°C of each other.

Use the TM calculator on neb.com to determine the appropriate annealing temperature for your specific polymerase and buffer composition. Recalculate this temperature when switching to a new enzyme, even if using the same primers with a different polymerase previously.

Calculate the GC content of your target sequence. For GC-rich amplicons, select a polymerase specifically designed for high-GC PCR such as OneAce or Q5 DNA polymerases, and follow the usage recommendations for any included additives.

Prepare PCR reactions on ice using chilled components and add them to a preheated thermocycler set to your denaturation temperature. Alternatively, use a hot-start enzyme like Q5 or OneTac hot-start DNA polymerase for convenient room-temperature setup with increased specificity.