i am facing problem in real time pcr optimization, i am using Bio-Rad system CFX96. i got different peaks on screen. but could not got sigmoid curve. when i run these samples on gel i got a clear band. whats wrong?? i think probe is not binding? i used probe without dilution..and i used primer conc 10pmole. plz suggest...see file attached
Dear Ahmed,
What are you using for a dye? SYNER Green or a TaqMan probe, or similar? This is important to know before being able to answer anythig. From the RFU scale you receive it looks that what you get is only background noise and no signal. Also it is important what is the amount of cDNA you use as well as the Taq-Polymerase mix and enzyme as some start already to be active before the annealing temperature.
Hi Rais,
I have a couple of questions ...
1. Are you sure your primers are validated? Do you obtain a single melting curve of your product?
2. Do you use Sybergreen? Also is this happening only with one gene or any gene tested?
Regards,
Dear Ahmed,
To be able to reply your question, actually one needs to know which dye you used. As it makes a lot of difference if it is SYBR green or TaqMan Probes or similar. From the graph you attached, the RFU are very low and therefore one can assume it is only a background noise you see in there and no signal. Nevertheless, is is also important to know the amount of cDNA you use as well as the TaqPolymerase (some already start to work before the annealing temperature is set so you get a lot of junck too) and it's mix as all these can make also a great deal of effects on the results.
i am using Taqman probe with FAM attached as flourophore and BHQ1 act as quincher. primers are specific. i got Tm of primers by using touch downPCR and gradient PCR and it was found 54C. in real time pcr, my setting were: 95C for 2min, 95C for 15 sec and 60C for 30 sec followed by 40 cycles. but i could not get the exact peak. when i run amplicon on 1.5% agarose gel then i got clear band. what is happening? the exact peak like sigmoid curve i could not achieve...plz suggest
The fluoresence range look too low (like noise in a blank) as if it was the default setting. There might be different setting in the machine to choose the FAM filter. otherwise I would check because there could be no filter installed.
Did you try to measure the baseline fluorescence before starting the PCR run? If you already have high signal from the TaqMan probes than the probe with the FAM and quencher is not well designed. Sometimes also the quencher does not quench well the FAM signal; in this case a different quencher might help as well. All these may cause your run not to get a logarithmic run and a lot of background. In these cases, you will need a new probe for the primer pair you have, or design all (primers and probe) anew.
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