PCR efficiencies - (Jan/29/2009 )

maybe a silly question, but is it worth busting my gut to get PCR efficiencies (calculated from standard curves) to be close as possible to -3.23?
at the moment i have 4 primer pairs with slopes of -2.99 to -3.07 (112-116% efficient). Since they are all similar, i can use 2-ddCt method right?

It is my understanding that efficiencies should be between 90-110%, so you are barely out of the range, but still, these don't look like they are performing well. Did you run melt curve analysis to see if amplification is specific? Also, sometimes the dilution factor that you choose will effect how the efficiency curves look. If you are doing extremes, such as 10-fold dilutions or 2-fold dilutions, you sometimes get too much of a spread of concentrations or too little of a spread. I go with 3- or 4-fold dilutions, especially if the primers look like they are close to passing efficiency tests.

I'm not sure if you can say that the primers are still ok to use even if they pass the equal efficiency test when they are still outside the acceptable range of individual efficiency. If it were me I would test alternative primers to see if you can't improve the efficiency.

Like grassgirl said, make sure you only measure your efficiencies for a standard curve that will be amplifying within your linear dynamic range, i.e. only use the dilutions that are going to reflect the actual amount of template you will be working with. So, I had a 116% primer, but I had calculated this over a HUGE range of starting template; I actually only needed a very small window of template concentrations, so what I ended up doing was going back to the standard curve and only using those 6 values that were within the range I was going to be looking at/encountering in my sample. I ended up with a better efficiency then.

Of course, this will only work if you have a really good idea of how much template you will be working with in your sample; if you don't then the linear range will have to be bigger to ensure you cover all possible starting concentrations.

Again, it is helpful to see if your primers are making absolutely pure product in the melt curve; that you aren't getting primer dimers, which will give you a greater efficiency than is really true.

Did you try to optimize you primer concentrations?

Mulletman on Jan 29 2009, 05:33 AM said:

You can still use ddCt method, IF you can sure all wells run in similar efficient every time (no more than 10% difference between largest and smallest, 10% is the standard my boss set). LinRegPCR can help you estimate the efficiency of each well.

Otherwise, you need to clean up or optimize your reaction.

As I have been told, the efficiency more than 110% means there is PCR inhibitor(s) in your reaction; and below 90% means the PCR condition is NOT optimized.
According to your information, you might need to clean up your reaction.