Hi,

For typical conditions, ideal amplicon length should be in between 70 and 200 bp. In your case, the product size is 77 basepairs.

There is a correlation between the amplicon length and real-time PCR results. You can read it in : Debode, F., Marien, A., Janssen, É., Bragard, C., & Berben, G. (2017). Influence of the amplicon length on real-time PCR results. Biotechnologie, Agronomie, Société et Environnement, 21(1), 3-11.

But the size of the amplicon is not the only cause for the failure of any realtime PCR reaction. Primer and probe design criteria are also one of the most important part one must look into. For example, you can chose primers which spans exon-exon junctions which will minimize the genomic DNA amplification or by avoiding the repeated sequences. Furthermore, SNPs which are underlying the 3' primer end are also known to have an impact on amplification.

Furthermore, you can refer to “Steps for a successful qPCR experiment” by IDT.

Thank you.

Hi,

if you are using Tamra as quencher this can be more challenging but still all normal Probe qPCR reagents should be able to get this done. Is your target high in GC or so? In adidtion are you sure your samples do contain the target sequence? You could run a gradient to see if your annealing temperature is not optimal in first instance and check if the primers are detecting your GOI e.b. by Sbr based qPCR

Good luck

Sven

What do you mean with "I do not get any results" ?

i) no product at all (no bad in a gel),

ii) just unspecific products (bands in a gel, but not at the right size)

iii) specific product (right band in a gel) but no amplification curves

iv) specific product but creepy amplification curves, no useful Ct values

v) something else I did not list: what, precisely?

Do positive controls work?

What probe system are you using (e.g. hydrolysis, hybridization, beacons, scorpion primers, ...)?

Hi

Agree with , Madhab Kumar Sen