Dear All,

I am hoping to sequence microbial DNA from human tissue samples. I have Qubited my samples and their concentrations are low, but there.

I have been having trouble getting "beautiful" bands after my PCR amplifications.

I use Walters et al., 2015 protocol re: primers + PCR programme. I have tested several DNA Polymerases (Sigma Taq, REDTaq, Platinum Hot Start, Q5). The first three produce great and bright single bands. However, there is a faint band in the negatives (the reaction mix + Molecular Grade Water). Q5 has no false positives in the PCR negative (the reaction mix + Molecular Grade Water), but the template bands are smeary and have non-specific bands (some bigger, some smaller) that do not match my positive controls. The exact same starting material has been used - this is not due to different reagent batches.

I believe that the "contamination" in my negative controls for the so to speak cheaper DNA Polymerases is due to manufacturing remnants (I have tested the samples, I work in a clean hood, use filtered tips, aliquots of clean reagents, everything is bleached and UVed etc).

I have already played around with the annealing and elongation step temperatures and times. I have changed the amount of DNA Polymerase, primers, and dNTPs going into the reaction.

In theory, I could proceed with one of the cheaper DNA Polymerases and do a post-sequencing clean-up re: the results found in my negatives. However, I would love to find a way around this, i.e. get beautiful bright single bands and nothing in my negatives. All ideas are welcome!!! Thanks!