too high PCR efficiency trouble

[Petrusha]

Hello,
I´ve just started with qPCR technique and need help.
My standard curve (5 samples of 10xdilutions plasmid with our insert) shows a slope around -2.55 that would mean PCR efficiency about 150%, sometimes even more. I know that only single product is amplified (agarose gel and HRM OK). Even I tried delete some dilutions from the curve, it did not help. I did not use touchdown technique (I´ve heard this could slightly overestimate efficiency when using Sybr). I´ve tried the same thing with 4 different inserts and still have the same problem-too high efficiency=too small difference beetween 2 consecutive dilutions. R value is 0.99 (OK).
The same problem with Sybr and TaqMan.
Any suggestions?
Thanks

[cds79]

Hi Petrusha,

have you tried to clean your cDNA before running your qPCR. It sounds like you may have some PCR inhibitor in your reaction.

[cds79]

Hi Petrusha,

have you tried to clean your cDNA before running your qPCR. It sounds like you may have some PCR inhibitor in your reaction.

[Petrusha]

Yes, I purified my plasmid DNA (Aratio is 2) before running qPCR.

[cds79]

how did you purified it?

[Petrusha]

It was commercial kit for plasmid purification (based on silica). Do you have your reliable plasmid purification method to recommend?

[cds79]

i have never worked with plasmids on qPCR, but i can tell you that with with the tissues samples we work with, we have found a terrific improvement of efficiencies after cleaning the cDNA with a columns. We have used Nucway spin column from ambion, but i think you may find also some other available.

Maybe you are carrying some ethanol or other chemicals from your purification, that's why you efficiencies are higher than 100%.

The other option is you primer design. Even though bad design results generally in efficiencies lower than 90%. Just have a look at it as well.