I designed a primer and probe for real time PCR using (https://www.eurofinsgenomics.eu/en/ecom/tools/qpcr-assay-design/) this website with the bp size shows 143. after i received the primer i did conventional gradient PCR to confirm the presence of band. but i got band between 400 to 500 instead 143. then i come to know about in-silico pcr tool for band visualization (http://genome.ucsc.edu). that shows my primer is the size of 409bp.
is it feasible to carried out with 409 bp for real time pcr?
Hi Siva,
The ideal amplicon size is usually between 70 and 200 bp, I am assuming you did your optimization using cDNA. Keeping the product size in this range ensures high primer efficiency and better overall real-time quality/resolution. Maybe redesign your primers? But if this is not an option then adjust cycling conditions to include longer extension times and give it a shot.
Hi, 409bp is quite big for qPCR. Try to re-design a primer with smaller amplicon size.
As Francis and Zx said, 400 bp is not an optimum qPCR amplicon size. If you the enough resources and time, try to redesign your primers, at least one of them, so you can get an amplification product around 70-170 bp. A bigger amplicon may impact your experiment by dampering the PCR efficiency, making it much harder for you to obtain a proper standard curve (~ -3.32), and, probably, you won' t be able to get a single peak melting Curve while determining melting point.
Sear friends I can only add that in my practice the best amplicon size is 65-70 bp. I.e. as short as it possible. MGB-probes can help you in this.
No, your product is too big. Design more primers and try again.
If that's the only primer that will give a product, you could test out it's efficiency, but I would predict the efficiency to be too low to be usable.
Good luck!
i am curious to understand why first you thought the product was 143 and then in turned to be 400 in reality. Are you sure this 400 bp fragment really corresponds to the gene you wanna amplify? Might it be an unspecific product according to my understanding because maybe these primers you designed were not specific enough and then you end up amplifying a 400 bp band.
If you will be designing new primers you should consider making primers for the fragment you already has for nested pcr. You wont have any nonspecific products.
Its too long. Its better to follow the range between 100-150 bp.
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