Hi all,
I'm trying to analyse bacterial diversity by amplifying 16S rRNA using primers targeting V3 and V4 region. The samples are human biopsy tissues.
After extracting the biopsy sample for total gDNA using the MoBio PowerSoil DNA extraction kit, the extracted DNA was used as PCR template, however I'm getting a very faint smear as the product migrates, what is causing this and has anyone else seen the same observation?
I've diluted my template and primer using EB buffer from QIAGEN. Does additional tris have an effect on PCR?
I'm also getting a larger and more intense smear for some prep, I suspect those are amplification when priming to the human origin?
The last 2 lanes from the right are positive control (Pseudomonas sp.) and negative control (10 mM Tris) respectively,