I am doing ddPCR for HIV targets (env, gag/LTR and 5'pol) with infected PBMCs. I am using uninfected PBMCs, SUPT1 cell lines as a negative control. I suddenly started having signal in my negative controls. I changed all my reagents, my bench and pipettes and even droplet reading oil. I repeated the experiment 4 times with the same signal in all my negative (bearing in mind that all the different controls are extracted at different times so it can't be an extraction contamination+ they were run through ddPCR before and were negative). I am not sure what could be plausible explanations other than a contamination in my samples/reagents?
P.S: the QX 200 ddPCR machine recently broke down and we fixed and serviced it (I don't know if that's related but mentioning it anyway).
HI, DD PCR is extremely sensitive . Hence, we can get cross contamination easily. The best way is to change the design of primers and probes itself, if contamination problem persist. Before that, we need to do cleanup of all equipments. Better inform the company person about sterilization, verification and validation of the machine, QX200. In short, do calibration again.
Then, add atleast two NTC, two NC to rule out the contamination. If possible repeat the assay with another person, on another day. Then, you can find out the reason easily. always keep the reagents, at minimal aliquots.
Hope, it will help you. If possible, share the graph here
thank you
Dr.Kalyanaraman
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