Does the amount of Internal Control to be spiked into the lysis buffer or lysate depend on the elution volume only?
Principally, yes. However, additional factors influencing the CT are:
1. Extraction efficiency, which depends on extraction method and sample material.
2. Volume of the eluate used for PCR.
Examples: Use of the Internal Control according to handbook instructions (0.1µl per 1µl elution volume) for two different workflows.
- Workflow 1: High extraction efficiency: use of 5 µl of the eluate for PCR results in a CT value of 29-30.
- Workflow 2: High extraction efficiency: use of 10 µl of the eluate for PCR results in a CT of 28-29.
Depending on the workflow, the amount of Internal Control to be added to the extraction might have to be adjusted to more than 0.1 µl per 1 µl elution volume, or to less than 0.1 µl per 1 µl elution volume, in order to achieve a CT value within the expected range.
Running the Internal Control DNA/RNA provided with the QuantiFast Pathogen +IC Kit in a separate reaction can serve as a reference for adjusting the amount of Internal Control DNA/RNA (High conc.) to be added to the extraction.