I am a beginner with PCR and working with mitochondrial primers and insects. In particular, I have created primers (several pairs) to amplify aphid DNA fragments from the COI and cytochrome b genes in the range of 150-250bp. I have had to switch primers for other reasons, and I am now inconsistently getting bands of the same size as my target DNA in my negative controls, which has been going on for several PCRs now with several different primer pairs.
I thought it was me (and still could be), but Iíve switched primers, switched kits, used other polymerases, and the band seems to appear at least every few PCRs. Iíve switched pipettes, UV radiated my pipettes, changed water, autoclaved my tubes and racks, ordered new primer stocks, but it keeps coming back. I also load my negative control first in a separate tube and set it aside before loading any of my positives. Iím running 35-40 cycles (need several cycles because the DNA is in low concentrations in my samples and partially degraded), using 1.5% agarose gels, and post-staining them. I thought this might be from carry over contamination when loading gels, but I have loaded my negative controls far away from other wells and same problem.
Iím still wondering if it is from gel loading. I didnít have any contamination for about 30 PCRs (several in another lab), and now I canít seem to get away from it. The bands are sometimes very bright and sometimes faint.
Any advice would be great because this is very frustrating and a huge waste of time. I have no problem amplifying my positive control; itís just the contamination issue.