Problem with TaqMan qPCR efficiency - (Aug/14/2016 )

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.

I'm no expert on this but I can see two potential issues:

1) The new primers may be defective. When you get ordinary primers they are a mix of all the different length products that were used to prepare the full-length primer, sometimes there are faults in this process that mean you receive a primer or two that consists mostly of short products. I would run a regular PCR and see how well these amplify.

2)Don't autoclave the water - autoclaves are used for sterilizing, and will certainly contain more impurities than your ultrapure water source (think where the steam comes from that makes the autoclave work...) If you are worried about sterility of ultrapure water, pass it through a 0.1 um filter.