I currently work on arbuscular mycorrizal fungi (AMF) that inhabit plant roots. Because I have very few roots to extract DNA from, I obtain a low DNA concentration after the extraction (beadbeating + DNeasy plant kit from Qiagen). Moreover, my guess is that plant DNA is much more abundant than fungal DNA.
I have tried running PCRs to amplify part of the fungal 18SrDNA (800bp) using primers that are AMF-specific. I used 1, 5 and 10 ul of DNA extracts (more or less corresponding to DNA amounts between 5 and 150 ng) with 0.2mM each dNTP and 0.2uM each primer. No amplification. In another PCR, I doubled the dNTPs and primers concentration. Still nothing.
I am just thinking that the target DNA might be too rare in the DNA extract so I might need to use nested PCR. The issue is that I study AMF communities using T-RFLP. With T-RFLP, the peak area of each T-RF is considered an estimate of the abundance of the T-RF (or OTU). I am wotrying that this assumption might not be valid anymore if I use nested PCR.
What so you think ? Can I still consider T-RFLP as quantitative after nested PCR ?