Hi, I'm doing doing RT-PCR to detect the presence (or not) of a pathogen. I've done it 6 six without any problems, however, my last three attempts failed, because I don't get a standard curve anymore.
The samples and the standards start in a low concentration, then they are simply not detected anymore (there is a gap in the graphic), and in the end they are detected again, but no signal of the standard curve.
I've already changed the standard dillutions (0,1; 1; 10; 100), the probe and the TaqMan, but nothing worked.
Is it possible that I'm mishandling it or it is likely a problem from one of the reagents?
Thanks!
This might be typically a problem of a wrong base line correction and a very low signal after amplification. As you can see the baseline fluorescence at cycle 1 is higher than yor plateau. This must differ at least one log scale; the plateau being 1 log higher. My first advice with respect to the baseline would be to set the cycles for baseline correction manually after e.g. cycle 6-8, or even later.
However, if you say that this is your normal control something must be really wrong. These curves really look like 'no amplification'. But first try to 'play' with the baseline correction. If this will not ameliorate your curves, you must indeed think of a damaged positive control (standard). The moreover you did change primers and probes. This can be simply tested I hope.
The other problem might be that your Taq polymerase is damaged; but I think thats unlikely.
So, one technical question. What is the signal and how does the amplification curve of your internal amplification (process) control looks like, or don't you use an internal control in you standards? I would strongly advice this in any case.
firstly clean all working area. change the PCR stock reagent to new one.
All good suggestions - but also look at the macro you used to calculate your standard curve. Has it been altered or corrupted in any way?
dear all Carl Gregory , Jack Greenhouse and Ashraf Abbas , thank you for your feedback. I attached the result I get from the qPCR. Indeed, the standard controls have been many times frozen and unfrozen, but in my last attempt, I used new ones, so it may not be from the standard controls. I was using a new primer stock for the three attempts, so I thought it could be from it, either from the dillution or from the primer itself (it is the same we always use, but a newly opened tube)
This might be typically a problem of a wrong base line correction and a very low signal after amplification. As you can see the baseline fluorescence at cycle 1 is higher than yor plateau. This must differ at least one log scale; the plateau being 1 log higher. My first advice with respect to the baseline would be to set the cycles for baseline correction manually after e.g. cycle 6-8, or even later.
However, if you say that this is your normal control something must be really wrong. These curves really look like 'no amplification'. But first try to 'play' with the baseline correction. If this will not ameliorate your curves, you must indeed think of a damaged positive control (standard). The moreover you did change primers and probes. This can be simply tested I hope.
The other problem might be that your Taq polymerase is damaged; but I think thats unlikely.
So, one technical question. What is the signal and how does the amplification curve of your internal amplification (process) control looks like, or don't you use an internal control in you standards? I would strongly advice this in any case.
Elizabeth Van Pelt-verkuil thanks for your answer! Usually, the amplification of the standards occur at cycle 30-36, but we do not use internal positive controls, only negative control.
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