| | Re: non replicable Ct number and a lot of other problems
I stopped working on real-time PCR. For now...maybe I will go back to it later. But we found out one or two things and I thought it could help other.
Since I worked this someone else tested primers, PCRmix, probe, programme. Only me the pipetter, and the sample should have been the problems. Finally I got triplicates together and assumed my pipetting was ok. I still got slope around 4 or worse instead of -3.3. And I decided it could not be pipetting (triplicates together). I was probably wrong.
A technician of my lab who is experienced and is suppose to pipette properly tryed it with the same weird results and bad efficiency. Finally she ordered a new pipette: 0.1 - 2 µL. She tryed twice before going on hollidays and got slope once 3.3 and once 3.6 I think. So maybe it is now really working... And the problem was pipetting. It was only on standard curve, so we will check later if is consistent or not.
Apparently reprodicible pipetting is not as easy as it seem to be. Good luck with your Real-time PCR, my "normal" PCR are making me crazy ^^