I have low efficiency problem with qPCR. My primers were designed based on the varible regions of 16S rRNA gene as I want to know the exact copy numbers of 16S rRNA gene. My standard curve was made from plasmid inserted with my target. The target fragment was cloned using topo TA cloning kit for sequecing. The plasmid was extracted and digested by restriction enzyme Pst I and the band was cutted from gel. I have changed thermal protocol and changed another pair of primers. The efficiency is still 60%-70%. The reaction components per 20 μL were 7 μL H2O, 10 μL 2x Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent Technologies) and 1 μL 10 μM of each primer.
I just hope whether your science support could help me figure out the problem, as you are the expert of qPCR.
Many thanks and look forward to your reply.