I have four genes of interest for a specific disease. There are total 8 different types of primers (4 forward and 4 reverse) in the multiplex system. Now, one primer's melting temperature is 58 degree C (which is the lowest among all) and the highest is 68 degree C. Now, what should be my annealing temperature? How much difference should be between annealing temperature and the lowest melting temperature of the primer?
Paul Rutland
The annealing temperature is usually 3-5c below the melting temperature of the primer but salt concentrations and enzyme type can make this very variable so running a gradient pcr from 53 to 63c would be the best way to get an answer to this problem
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