By the limitation of the region of interest for my research I have the only available primer design option that returns 26 bp amplicon length. Yes,, amplicon, not primers :)
So, will it work?:)
Your opinions and experience are very valuable!
"Amplicon length" usually applies to the full length of a PCR product, and that means it includes the primer sequences. If that is what YOU mean by amplicon length, then it seems certain that your primers must actually overlap and, in that case, no, all you will get is primer dimer. On the other hand, if what YOU mean by amplicon length is actually the length between the 3' ends of the left and right primers, then the answer is yes, this PCR could possibly work and the actual "amplicon length" will be 26 plus the length of the primers or around 66 bp. I have used such short amplicons occasionally for the amplification of GC rich regions and, as long as they are specific and efficient, they will work OK for SYBR green qPCR.
Dear Ruslan
I hope this link will be useful for your enquery
https://www.gene-quantification.de/national-measurement-system-qpcr-guide.pdf
Regards
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