Did you try to produce a standard curve with the obtained results from these 3 points? The slope of standard curve indicates PCR efficiency. The efficiency of the PCR should be between 90–100% (−3.6 ≥ slope ≥ −3.3). If the slope is optimal, then you have a restricted detection limit If the slope is below –3.6, then the PCR has poor efficiency. This poor efficiency could be due to:

1) Inaccurate sample and reagent pipetting

2) PCR inhibitors in your sample (you can check it by UV spectrophotometer – check your A260/A280 ratio)

3) Your PCR primer and/or probe design may not be optimal

Generally I make 2 or three different set of primers, I make melting curves for these primers. I chose the primer set which gave me best melting curve. For no reason, some set of primers do not give good melting curve, I discard them.

Dear Mahyar

Use fresh diluted pcr product for prudiucing curve. Extracted DNA is not suitable for this reason especialy when your gene is not linear ones. Mitochondrial genes, circular ones I mean have a negative effect on PCR

efficacy.

Dear All,

Thank you very much for your answers and good recommendations.

We are going to do some changes in our protocols and lets see what will be happened.

dear All,

same problem: The efficiency and slope are perfect for all of three points but when you add fourth and fifth points efficiency decreased and slope goes off. who knows its reason might be?

thank you.

Dear Mahyar

would you attached a photo of your last try?

Dear Mohsen,

The photos are attached.

Dear Mahyar

First of all again I emphasise you should use the fresh pcr product for drawing your standard curve

the other important note that I find of your curve is that you have a problem in your sampling stage so you should use a calibrate sampler for this issue. Change your sampler by a new ones the other important note is Wright handling of sampler. In final I suggest using a trublshoting instructions for drawing St curve that present on line. You should improve your sampling stage by several time. May be more than 10 time to reach a

good St curve. Do not disappear an countinue as I advised.

BEST ...

Dear Mohsen,

Thank you very much.

Dear Seyed, reading the replies to your question, I wonder if the problem is the set of primers and probe. If you have used fresh reagents and template extractions, and your preparation techniques are accurate (i.e. the dilutions are correct), the assay might be telling you the truth and the linearity observed with the three points might be the limit. As several answers point out, there are several factors that still need to be checked to rule out possible errors. Nonetheless, I must point out that if you are trying to adapt an assay to your needs, I suggest to follow the directions as stated in the publication, in my experience, the quality of the reagents can have profound effects on the downstream results.