I have run a real-time PCR reaction with SYBR and included the following controls
1. Positive control: all the reactions mix with DNA from a target organism (V. cholerae)
2. Negative control: all the reactions mix with DNA from non-target organism (E. coli)
3. No template control: all the rreaction mix without DNA
I constantly get the same melt peaks as the positive in my negative control. I have also run gels and get the same band as my positive contro in my negative.
I have used this primers before and tested their specificity against different organisms including E. coli and they showed to be specific. I have tryed changing everything, meaning new primers, muster mix, water, wiped the bench with bleach but I still get contamination