How do I choose primers for PCR?

Posted June 22, 2020

Polymerase Chain Reaction (PCR)

Answer

Primers are necessary when running a PCR reaction, which should generally have the following properties:

Length of 18-24 bases.
40-60% G/C content.
Start and end with 1-2 G/C pairs.
Melting temperature (Tm) of 50-60°C.
Primer pairs should have similar Tm, within 5°C of each other.
Primer pairs should not have complementary regions.

Additional resources

6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*

Chen, S. C., Halliday, C. L., & Meyer, W. (2002). A review of nucleic acid-based diagnostic tests for systemic mycoses with an emphasis on polymerase chain reaction-based assays. Medical mycology, 40(4), 333-357.

Chuang, L. Y., Cheng, Y. H., & Yang, C. H. (2013). Specific primer design for the polymerase chain reaction. Biotechnology letters, 35(10), 1541-1549.

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