The primers are designed for RT PCR. Is it possible to use the same for conventional PCR?
It will depend on the purpose of your study, but if the designed primers are specific to your target, the amplicon size(s) can be separated on gel, and it is not a probe-based qPCR system like Taqman, then it should work in a conventional PCR as well.
Hello Suganthi,
sure they can: the principle is identical. The only difference between standard PCR and the most common Q-PCR techniques is than in Q-PCR you either add an intercalating agent such as SyBr Green to detect the production of double stranded DNA or you use a TaqMan probe binding in the target region which is degraded during amplification by the 5'-3'-exonuclease activity of the Taq polymerase. This causes physical separation of the fluorophore from the quencher and emission of fluorescence. That said, amplification works in Q-PCR exactly as in normal PCR. Typical TaqMan primers amplify very small regions, often less than 100bp. SyBr Green primers are often designed to amplfy slightly larger regions, often in the 100-300bp range.
for the conventional/ semi-q PCR you can use the same primers in fact it is better to first standardize the annealing temperature of your primers with semi-q PCR before going for Real-time it saves you plenty of reagents! All the best!
Hello Suganthi,
I fully agree with Rocco. However, since you check your amplicon on agarose gel for conventional PCR, there is no need to include the TaqMan probe or adding the intercaling agent when you prepare the PCR mix.
All the best.
Possible but as real-time products are restricted in size, amplicons can be difficult to distinguish from primer dimer
Hi everybody
That was my important question
Thank you all, specially Jacques Davy Ibaba.
hi Dr. Suganthi
there is no different. only different between the two process is in the detection chemistry which found only in real time pcr because real time pcr is in its step is pcr put detected through the progress pf the reaction not at the end of it like normal pcr
Thank you all for your answers
I am new in the field and these help so much.
However I would still like to ask a question.
I have designed primers for qpcr and already run it
The following step is cloning
I would like to know if I can use the same primers for normal pcr before cloning
Thanks
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