I am using real-time PCR to amplify regions of my cDNA using primers designed based on the Salmonella genome. I am using a bunch of primers and for some of them, I keep getting contamination in my NTCs. I've replaced the Mastermix, ordered new primers, changed the water and ordered expensive ambion water but to no avail.
It was suggested that it is caused by contamination in the SYBR Green from DNA left from the E.coli that was used to produce the polymerase. So we treated the SYBR Green with AluI and DNases but there is still amplification in my NTCs.
Beyond redisigning the primers, or using probes etc, is there anything else I can try? I checked annealing optimization for the primers and they work as high as 63 degrees with no primer dimer on conventional PCR. So I used this high annealing temp in the real-time run in the hope for high specificity for my target but still get NTC contamination.
Any help much appreciated!