Does anyone know if a pair of primers designed to use with fluorescent probes (using TaqMan system) could be used in a system that do not use probes (like EvaGreen), obtaining the same results?
You could try, the PCR would work. But you need to check that you have a unique PCR product.
The results will not be exactly the same , it is not the same technology nor the same enzyme for PCR.
You can use either EvaGreen or SYBR green but you will have to follow melting cure after PCR. Melting curve will be will indicate the contribution of non specific product or primer dimer. Some companies will provide you with 2X master mix which work fine but it again depend on your primer design.
used for what? Detection or quantitation? Detection is OK, if you have figured out which is the smallest DNA concentration which gives a reliable and true amplification (to avoid false detection of primer dimers, etc as PCR products and melting curve analysis, which could be really slow on some instruments). Checking by electrophoresis is better than melting curve analysis IHO.
For quantitation purposes commericial mixtures with intercalating dyes could be a challenge, mostly because you dont know the additives (BSA, Betaine, etc.) and Mg concentrations in the mixture. The efficiency of of your reactions and the the inhibitory effect of some compounds in your isolated DNA may be different in the two reactions... but you should check.... If I were you, i wouldnt switch from taqman to sybrgreen, , i use sybrgreen just for preliminary experiments, or when im lazy or poor.... good luck
Appreciate all answers, thanks a lot. I will use the EvaGreen for detection at the first moment and if I detect my target I might need to go a step towards quantification. To calibrate the system I can use samples with known number of parasites and check the accuracy.
HI Francisco, is a good approach for testing purposes. I do it usually. In some cases you can use the set of primers without the probe specially if you are doing PCR with simple samples or pure cultures. If you are obtaining certain level of primer dimer, you can add one simple modification to your PCR protocol. Include one Fl acquisition point ( 5s) in a temperature near to melting point of your procedure.
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