Dear all, in a PCR (conventional & realtime both), if we achieve our desired product (GOI) and along with, there is also no primer dimer (PD) formation seen on the gel and melt curve, does this necessarily indicate that all of the primers (forward & reverse both) of a certain reaction, have literally consumed into this reaction for constructing the required products / amplicons OR still some leftover primers are there in that reaction after ending up all the PCR cycles?
Is there any statistical approach or formula exists to know about any possible probability or even the percentage of leftover primers' strands per PCR reaction? These primers are always in known concentrations and volumes, so why not we can have some estimation of the remaining unused ones?
Sincerely......
If you did come close to using up all of your primers, the final rounds would have far more PCR product than primer in them and most of the PCR product when it became single-stranded during melting temperature would not get a primer bound for extension.
In my experience, when I did too many rounds of PCR the nice product band that was present at 18 to 25 rounds of replication completely disappeared by 30 rounds, sometimes with a higher molecular weight smear visible and sometimes with nothing of note visible.
hi
optimization of primers concentration on different temperature gradient is an essential step , even though the guarantee of certain company
If you did come close to using up all of your primers, the final rounds would have far more PCR product than primer in them and most of the PCR product when it became single-stranded during melting temperature would not get a primer bound for extension.
In my experience, when I did too many rounds of PCR the nice product band that was present at 18 to 25 rounds of replication completely disappeared by 30 rounds, sometimes with a higher molecular weight smear visible and sometimes with nothing of note visible.
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