There is no need to run the PCR for more cycles than required to get to the plateau phase. If the sample with the lowest concentration reaches the plateau after 30 cycles - then 30 cycles would be sufficient.

There is actually never a good reason to run the PCR for more than 40 cycles at all. If you have a single molecule in the reaction (and less is impossible!) you will get the plateau after about 37-40 cycles (given the reaction is well optimized).

Doing more cycles than neccesary is not a problem, but it is a waste of time.*

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*There is one possible exception: if all your samples have low ct values (say at about 20 cycles) and check for specific amplification (what you should always do), it may be that unspecific products are amplified in later cycles (say, after 30 cycles) . These unspecific amplifications would not impact the ct values of your samples. Analyzing the products after 50 cycles won't tell you if these unspecific products can be a problem or not. If you had stopped the PCR after 30 or 35 cycles, you could see if these products are already  detectable at earier cycles that may be relevant for quantification.**

** Note that this is not a particular problem of SYBR Green - quantification using sequence specific probes are also affected by the amplification of unspecific products through a change in the amplification efficiency of the specific product! [-> competition effects]

There is no need to run the PCR for more cycles than required to get to the plateau phase. If the sample with the lowest concentration reaches the plateau after 30 cycles - then 30 cycles would be sufficient.

There is actually never a good reason to run the PCR for more than 40 cycles at all. If you have a single molecule in the reaction (and less is impossible!) you will get the plateau after about 37-40 cycles (given the reaction is well optimized).

Doing more cycles than neccesary is not a problem, but it is a waste of time.*

---

*There is one possible exception: if all your samples have low ct values (say at about 20 cycles) and check for specific amplification (what you should always do), it may be that unspecific products are amplified in later cycles (say, after 30 cycles) . These unspecific amplifications would not impact the ct values of your samples. Analyzing the products after 50 cycles won't tell you if these unspecific products can be a problem or not. If you had stopped the PCR after 30 or 35 cycles, you could see if these products are already  detectable at earier cycles that may be relevant for quantification.**

** Note that this is not a particular problem of SYBR Green - quantification using sequence specific probes are also affected by the amplification of unspecific products through a change in the amplification efficiency of the specific product! [-> competition effects]

I think that most experienced users of qPCR would say that you can't make reliable quantitative comparisons for samples with Ct values above the 30 to 33 range. So there is generally no value in running more than 40 cycles.  If you see a plateau after 40 cycles there are (at least) two possible concerns.  The first is that the product is not specific--after 50 cycles, you will almost always get some primer dimer or other artefactual product.  So you need to check whether the product is the correct one.   For Sybr green reactions, the melt curve will give you some indication, but you also need to run a gel or use some other test to determine the exact nature of the product.  Even if the product is specific, another possibility is that the efficiency of the qPCR reaction is very low.  In this case, since analysis of qPCR results usually assumes a relatively high reaction efficiency, any quantitative calculations that you make will not be accurate.

Dear Bushra,

All true. Additionally, in my opinion 35-40 cycles are sufficient. As you know, usually Ct higher than 30-35 is considered to be a false positive, so 35-40 cycles are sufficient. Also in qPCR Ct is important and plateau isn't very important.

Good luck,

The plateau is indirectly important to ensure having enough values in the exponential (log-linear) phase of the amplification curve - the part that is used to determine the ct value.

Thank you everyone for sharing the logical answers!

The question is if the amplification of the unspecific products impact the amplification of the specific product during the early cycles (before the Ct). This is what you would need to check. It's often simpler to test primers until you find a pair that does not give unspecific products.

Aside: many people say that sequence-specific detection (like with hydrolysis probes, hybridization probes, beacons etc.) is superior to dsDNA-specific dyes like SYBR Green for qPCR, just because such unspecific products are not detected. However, if they are generated and amplified, this may still interfere with the amplification (efficiency) of the specific product and thus bias the Ct value. So there is actually the drawback that I have to make gels to visualize the potential amplification of unspecific products. For the mere purpose of quantification, sequence-specific detection ha no advantage. The advantages are that you can more easily detect (not quantify!) the presence for even low amounts of the target sequence even in the presence of the amplification of unspecific products, without a need of any post-PCR processing, and that it is possible to do multiplex PCRs, which is but quite hard to get reliable quantifications with.