I am getting amplification and specific melt-curves in Non-Template Control sample (with water instead of cDNA). I ordered some primers and reconstituted them to 100 uM and diluted them to 10 uM inside LAF in NFW (nuclease-free water). At the first use, I got good amplification in the sample and no amplification at all in NTC (Checked on an agarose gel as well). When I used it the second time I got NTC in all samples. I freshly aliquoted 10 uM primers inside LLAF but I still got amplification in NTC. To confirm whether there is problem in NFW or SYBR, I did various reactions with only SYBR + primer, SYBR + NFW and SYBR + primer + NFW. I found amplification in whenever primer was added and no amplification in only SYBR + NFW. Hence I concluded that there is some contamination in primer stock only. But I seriously wonder why it happens that after opening just once and that even inside LAF, the primer stock becomes contaminated or there might be some issue with RT-PCR machine? Can anyone please suggest anything to troubleshoot this problem?

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