Hi everyone,
I’m doing SNP genotyping (forensic DNA phenotyping) using PACE (PCR Allele Competitive Extension) qPCR chemistry. I work with very low DNA input from Chelex extractions. Recently, my NTCs started amplifying on the allelic discrimination plots, but only after "recycling" the plate (running extra cycles to force the reaction).
I’ve systematically swapped out every single reagent and consumable to track it down:
• New PCR-grade water
• New master mix aliquot
• New SNP assay
• New PCR strips and Eppendorfs
The only variable I haven't changed is the pipette. Here is the kicker: I share this pipette with 5 other students who use it for human cell culture. I use filter tips, but I suspect the external surface or the ejector mechanism is loaded with DNA aerosols from their high-biomass work, physically dropping amplifiable DNA into my tubes.
To make things worse, our hood doesn't have UV, and we’ve historically only cleaned with 70% EtOH. The hood is acrylic, so I can't just soak it in 10% bleach without crazing the plastic.
Has anyone dealt with a similar contamination nightmare from shared cell culture equipment? Is there any specific test I can run to prove the source before I ask my PI for dedicated pipettes or decontaminating reagents?
Thanks.