I have some troubles with my TaqMan qPCR experiments.
Actually I have 2 primers (ITS1 and ITS2) and 1 probe that are supposed to amplify a specific region of my fungal DNA. The 5’ end of my probe is linked to Cy5 (red fluorophore).
What I am doing right now is to test the efficiency of this reaction (of my fluorescent probe). For this purpose, I have to do serial dilution of my target DNA (3ng/tube to 3x10^(-5) ng/tube) and to run qPCR separately (in order to create a standard curve).
2 weeks ago, I performed this experiment, and it seemed that my probe was functioning (see attached file). The reaction efficiency was above 0.90. Then last week I performed again the same experiment using newly ordered tubes of primers (ITS1 and ITS2), but it didn’t work perfectly for unknown reason.
I was wondering if someone could give me some advice on why my qPCR didn’t work perfectly as last time (see attached file). My R^2 value is 0.966, but the reaction efficiency is 0.424. I used double-autoclaved MilliQ water to dilute my primers and my probe. Is it possible that my water contained PCR inhibitors (but I autoclaved it for the third time before adding it to primer tubes)?
Some details about my experiment;
I am using Rotor-Gene Multiplex PCR Kit as my mastermix (stored at -20C). So what I am mixing is this mastermix, 2 primers, TaqMan probe, nuclease-free water, and fungal DNA.
For my second experiment, I used defrosted aliquot of probe, while I took the desired volume directly from the original tube for my first experiment.
The thermocycler I am using is Rotor Gene 6000 from Corbett (now Qiagen).
Thank you in advance for your help.