Call for help: Multiplex-strange foreign microsat only found in negative control

[cheeho0]

Hi to all. Hope some Masters could help me out with my problem which has taking place for 2-3 months.

I currenty conducting a research on a gecko species genetic variation using primers/markers which screened for a related species of mine (cross-amplification it is). For this while, I have been "haunted" with "contamination" alike problem. The NTC (negative control) in the multiplex (TouchDown PCR with 35 cycles, all primers are flourescent labelled primers; blue, yellow,red, green) seems keep showing band/s on and off. At first I thought the primer/s might be the cause as I had changed all reagants (always work on Lamina Flow, with autoclaved tips, water, etc). When I have genotyped the ntc, it shows there is a microsat peak/s (mostly the yellow labelled) in the NTC with size (157bp) that close to my wanted product size (160bp), for instance. But this microsat peak/s has never found in my other templates. This is very strange that if one or more of my primers are contaminated the contamination peak/s should have had appeared in my other template (because it's mutliplex) apart from my ntc AND other uncontaminated primers in the multiplex would have also amplified the contaminant eventually more different peaks of different coloured labelled would have showed up too. I have attached images for you to see.

I am now scratching my head still incomprehend what have had happened. Your opion and suggestion is desperately needed

Thanks a lot.