Primer efficiency decrease

[zera]

Hello,

I'm quite new to the qPCR world, and for the moment I can't say that I'm very successful...
My major problem at the moment is that I observed a big decrease in my primer efficiencies when I compare my last experiments with the experiments I performed last week. With the same primers, using the same template DNA, last week I had an efficiency of 1.9, and this week I can't go higher than 1.3... What could cause such a thing? I use the Roche LigthCycler system, with SybrGreen.
Thank you so much for your help!

[mchlbrmn]

Since no one else answers, I'll say, "I don't know". Were the primers stored frozen, and in buffer? They could degrade otherwise (although that doesn't seem very likely). I guess you also might see that if someone forget the enzyme mix out overnight, or an error was made in diluting the reaction mix, template, or primers.

[zera]

Thanks for your answer, even if I kind of guessed that no one had the perfect answer...
Considering that I have 5 primers pairs that I used at the moment, and that all of them showed a big decrease in efficiency, I doubt that it comes from the primers... I was suspecting something in the mix. For instance I was wondering about light sensitivity of the SybrGreen? when I had this problem, the sun was really shining, and I was not very careful about protecting my plate from the light (I have to go to another building to use the qPCR machine, so I was outside for maybe 2 minutes). Could that be a problem?

[mchlbrmn]

I don't think you should do your PCR at the beach. 2minutes? I don't know how much difference that would make. I suppose 2 minutes of direct sunlight might equal 1/2 hour or more of room light (just to pull supposed numbers out of nowhere). But that wouldn't actually affect the enzyme's efficiency, would it? I think it would weaken the fluorescence intensity, and push the Cp back, but it shouldn't change the spacing between dilutions, which is what the PCR efficiency is calculated from. You should be able to see if the height of the plateau is lower than usual to see if the SYBR got used up by the sunlight (unless the PCR machine software does stuff to stretch out a weak signal; I'm not a qPCR expert).

So that leaves the enzyme mix encountering a mishap in storage, or "pilot error" with the master mix (easy for me to say from here). You could double check to see if somehow your program got changed, and the incubation time reduced.

[zera]

Yes, I agree, what you say about sunlight make perfect sense...
I would say however that no, I can't think of any mistake made during storage (the mix gets only one thawing before being used) or when samples were preped, but I'm obviously wrong, as these low efficiency are due to something... I will check the program, even if we don't share programs...
Thank you for your help !

[mchlbrmn]

Hopefully it was a one time glitch, whatever caused it, and fresh reagents on a new day will work well.