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.
Not necessarily. Ideally, primers are single stranded short DNA segments. In PCR we generally use primers in excess. However, when the primers are not designed optimally (sometimes we have to ignore certain optimal primer parameters to suit our requirements) they may have some regions of complementary bases among themselves. These later form primer dimers of length that is roughly equal to the sum of the length of both the primers used in the PCR and show up in your gels.
When the primers are ideal, they will not form dimers and will remain single stranded of small length. Being smaller in length and single stranded, these will rapidly migrate through gels into the buffer and will not show up. Similarly, being single stranded they will not show up in your melting curves.
In your case, I think your primers are ideally designed, since they give good amplification of your GOI without any primer dimer. Else you would have seen dimers in the NTC too. But complete exhaustion of primers is seldom achieved and it is also difficult to ascertain the ratio of the unused fraction.
SD
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