Hello , it about my no template control (NTC) result for reference gene (GAPDH). I already run many times and use different batch of reference gene (GAPDH). I also change the master mix and nuclease free water ( using new). But when i run , it amplified.
It's probably residual contamination. Clean your work area (if you use bench paper change it out!), clean all your pipets, and use filter tips. Make sure no one else is using your water too! I had a lab mate "borrowing" my nuclease free QPR water and they contaminated it with gDNA. I suggest aliquoting and freezing your water, primers, etc. and using a fresh tube each time.
You have contamination. No point in figuring out exactly where it came from, just do a "toss everything, clean everything, and try again".
To add to the above: throw away ALL of your reagents (primers, buffers, water, etc.). Don't let anyone else use your micropipettes. And don't use the same micropipettes for handing amplified DNA (e.g. loading PCR into an agarose gel) that you use for setting up qPCR.
Make sure you are mixing your samples gently. Don't pipette up and down in the qPCR plate. That creates aerosols and can cross-contaminate your samples. Best way to mix is to put in all of the reagents, seal the plate, gently vortex, and spin down in a centrifuge to collect all of the sample in the bottom of the wells.
Good luck!
Its definitely contamination. Please use all new reagents including the primers, further I would suggest to use aerosol resistant tips and set up reaction in a biosafety hood.
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