I’ve been trying to calculate relative telomere length using Cawthon’s monochrome multiplex PCR method on a Bio-Rad CFX96™ Real-Time PCR Detection System. However, when I attempt to run the PCR, the cycles don’t initiate, and an error message appears. Interestingly, other protocols work perfectly fine on the same machine.

I’m strictly following the cycling conditions described in the original paper, but I still encounter this issue. Has anyone experienced a similar problem, or can suggest potential causes and solutions? Could it be related to the specific cycling parameters, reaction setup, or instrument settings?

Any advice would be greatly appreciated!