I am using the High Capacity cDNA Reverse Transcription Kit from Thermo Fisher and the GeneAmp® PCR System 9700 thermocycler to synthesise cDNA. The method specifies the following protocol:
Step 1: 25°C 10 minutes
Step 2: 37°C 120 minutes
Step 3: 85°C 5 minutes
Step 4: 4°C Hold
but does not mention the number of cycles. What number of cycles should I set on the thermocycler, considering it only accepts values between 2 and 99?
It is a single cycle, step 3 will inactivate the enzyme. As your thermocycler, you can set it to 2 cycles and end the program when the first is finished.
In general, cDNA synthesis is a one-cycle process. So, you might be able to try stopping the reaction after a single cycle.
One cycle is required for cDNA synthesis....if your thermal cycler does not accept 1 cycle, then set it at 2 and stop after end of one cycle. You can also call the application specialist to help you with the settings
RT as mentioned already by other colleagues is a single cycle divided into 3 main steps 1)denaturation and annealing; 2) DNA polymerization 3) Enzyme deactivation
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