PCR amplification of 16S rRNA gene failure from total soil DNA
I isolated the DNA using Epicenter (Soilmaster kit) and also MoBIO (Powersoil kit). The isolated DNA is pure as evident from the gel pic. The amplification was done varying different parameters like using fermentas taq pol, Phusion HF PCR master mix, changing the universal eubacterial primers from 1.5 kb size to 1 kb fragment size. The PCR conditions were also changed: increasing the denaturation temperature and time, changing the annealing temperature of the primers. The positive control DNA from a cultured bacteria showed sharp clear good amplification whereas the metagenomic DNA does not. I even made the PCR mixture separately rather than using just commercially available master mix. Used 1% BSA in the reaction mixture. Furthermore I quantified the isolated DNA which yielded (4760 ng/ml).
I don't know if this much concentration is way too little to be used as template in PCR. However, I used 1,2,3,4,5,6 microliter of the template DNA in PCR reaction, but could not get any amplification. Kindly help me on this and suggest alternatives.