Much shorter can be problematic as the specific product may not be distinguishable from primer-dimers.

Much longer may compromise the amplification efficiency, particularily when short cycling times are used. Reannealing and digestion by Exo+ or Endo+ polymerases will also negatively affect the (apparent) amplification efficiency.

As long as the amplification efficiency is not impaired, longer is better, because the signal yield (and, thus, the signal:noise ratio) increases with product length.This applies to SYBR Green (and other MGBs)  but not to sequence-specific probe systems.

Much shorter can be problematic as the specific product may not be distinguishable from primer-dimers.

Much longer may compromise the amplification efficiency, particularily when short cycling times are used. Reannealing and digestion by Exo+ or Endo+ polymerases will also negatively affect the (apparent) amplification efficiency.

As long as the amplification efficiency is not impaired, longer is better, because the signal yield (and, thus, the signal:noise ratio) increases with product length.This applies to SYBR Green (and other MGBs)  but not to sequence-specific probe systems.

Hi Jojo,

Amplicon length of 100 bp would work perfectly. I usually design primers that will amplify between 75-110 bp. I do not recommend amplicon length of 200-300 because this length may reduce the PCR efficiency, even though I often see that this length is recommended.

Good luck

Pre-made commercial qPCR mastermixes typically state a recommended range of amplicon lengths (usually

It is possible also to obtain amplicon of 75 bp length. But you need to melting curve analysis with your negatif control to distinguish your amplicon from primer dimers. 

As everyone else has said here, the reason for the optimal amplicon length is optimizing efficiency and accuracy. Too short and you will have a weird looking melt curve which can't distinguish from any primer dimers present and too long and the efficiency is compromised. The highest amplicon I have designed without compromising efficiency was 210bp. Usually I aim for 150 but this transcript was similar to other variants of this particular gene. 

Dear 100 bp gave you good results.

Hi, I am totally agree. 100bp it a good amplicon length to design primers. We always design primers between 70-150 bp, and a Tm about 70-72 ºC in oligo7 software. Thus, we  using an annealing temperature during the PCR at 68-70ºC (amplification at 72ºC). We have good results at this way.

I have successfully usws qPCR to amplify products from 80 - 300 bp. Longer products can still be amplified but, apart from the decrease in PCR efficiency, longer PCR products may result in melting curves with strange formats or even double peaks, since melting of the long amplicon may not occur at the same temperature. Differences in GC content along the strand may result in parts of amplicon being opened earlier than others. Shorter amplicons (

@Vlademir:

I am curious: have you analysed the dependency between amplicon length and amplification efficiency? Many years ago I did this for 3 genes, generating amplicons between 80-350 bp for each and I was not able to detect such a dependency.

Your point on the melting curves is good. However, it is not a problem für qPCR to have a "more complicated looking melting peak". It would be a problem for mutation detection or variant calling in high-resolution melting-curve analyses (but that's not qPCR, although it is performed using a qPCR instrument).

If you do two-fold dilution series of your template and see corresponding one-by-one increments in Ct value, then your qPCR is working efficiently. You CAN amplify longer templates with the usual efficiency given appropriate reaction conditions. For example, qPCR to quantify illumina sequencing libraries assume a 450bp template (see attached). 

I prefer < 100bp to a  maximum of 150.

Plz take a look at the following links:

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=14&cad=rja&uact=8&ved=2ahUKEwjgtJ2dmtXlAhUVThUIHUU8BVAQFjANegQIBhAC&url=https%3A%2F%2Fwww.gene-quantification.de%2Feurogentec-qPCR-guide.pdf&usg=AOvVaw3744RZiZfdUFjFmrZxHFRE

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=15&cad=rja&uact=8&ved=2ahUKEwjgtJ2dmtXlAhUVThUIHUU8BVAQFjAOegQIBxAC&url=https%3A%2F%2Fwww.gene-quantification.de%2Feurogentec-qq-troubleshooting.pdf&usg=AOvVaw2QRx6O_vk2sJVX3CoXiKvO

Dear Jochen Wilhelm

What do you mean by Exo+ or Endo+ polymerases?

Please clarify it to me.

Thanks

Polymerases having exo- or endonucleolytic activity (usually as part of a proof-reading activity)

https://en.wikipedia.org/wiki/Exonuclease

https://en.wikipedia.org/wiki/Proofreading_(biology)