I want to quantify the anammox bacteria in sludge samples. I have read few papers which use qPCR technique for quantification. However, most of the papers work in following sequence
1) Extraction of DNA
2) Amplification of target DNA with PCR
3) qPCR
The method also includes standard curve generation and cloning.
I am unable to understand the requirement of cloning in this process. Can the amplicons generated in PCR be directly used for qPCR.