Ur qpcr curves seem fine. As i see from your attachments, u have not set any threshold baseline.. set a threshold either automatically or manually. So that it lies at level of 0.4-0.5 of delta Rn because at that particular level, ur products have started to form (24-25cycles in X axis). Regarding, different heights of the three replicates in ur amplification curve, its simply because of manual error in pipetting that has caused formation of different amount of products in each well of triplicate. And so u get peaks at differnet level
Ur qpcr curves seem fine. As i see from your attachments, u have not set any threshold baseline.. set a threshold either automatically or manually. So that it lies at level of 0.4-0.5 of delta Rn because at that particular level, ur products have started to form (24-25cycles in X axis). Regarding, different heights of the three replicates in ur amplification curve, its simply because of manual error in pipetting that has caused formation of different amount of products in each well of triplicate. And so u get peaks at differnet level
This just looks like noise, combined with a rather high level of background fluorescence to me. It's worth remembering that logarithmic plots tend to amplify small variations around near-zero fluorescence. I suspect that if you did a linear plot, you would see nothing anomalous - though I prefer log plots for precisely this reason. And a Happy New Year to you all!
This just looks like noise, combined with a rather high level of background fluorescence to me. It's worth remembering that logarithmic plots tend to amplify small variations around near-zero fluorescence. I suspect that if you did a linear plot, you would see nothing anomalous - though I prefer log plots for precisely this reason. And a Happy New Year to you all!
As the others have already told you, It doesn't seem to strange to call our attention ... But you should give us some more info in order to let us evaluate If it is strange or not.
For instance:
What is the ratio of primer to probe in your reaction? Was it evaluated during your protocol optimization or did you use someone else1s protocol without changes? What qPCR equipment are you using? Could you send a pic of baselined and thresholded results or the experimental file to us? What reagents and reaction protocol are you using? How many cycles? DNA, cDNA concentration?
With these data we may evaluate a little bit better and give you our impressions about your results. Ok?
As the others have already told you, It doesn't seem to strange to call our attention ... But you should give us some more info in order to let us evaluate If it is strange or not.
For instance:
What is the ratio of primer to probe in your reaction? Was it evaluated during your protocol optimization or did you use someone else1s protocol without changes? What qPCR equipment are you using? Could you send a pic of baselined and thresholded results or the experimental file to us? What reagents and reaction protocol are you using? How many cycles? DNA, cDNA concentration?
With these data we may evaluate a little bit better and give you our impressions about your results. Ok?
Hi Subhasri Biswas , thanks for your answer. However, doesn't it seem strange that some curves do not follow a rectangular hyperbolic shape closely? I was wondering if (1) this is normal (2) what are the possible causes of these shapes.
This is another set of triplicates that I obtained recently. One of the curves does not follow a rectangular hyperbolic shape like the others.
Hi Subhasri Biswas , thanks for your answer. However, doesn't it seem strange that some curves do not follow a rectangular hyperbolic shape closely? I was wondering if (1) this is normal (2) what are the possible causes of these shapes.
This is another set of triplicates that I obtained recently. One of the curves does not follow a rectangular hyperbolic shape like the others.
Hi there! I don't know if you solved this already, but just in case I write. I had recently this problem and came to this post to try to find a solution, I found it elsewhere in the end, so I am writing for all the future newcomers as well. The problem with this is that the machine is reading the ROX value on your endogenous control. If you didn't add ROX to your samples (like me), this makes the machine subtract the values of your endogenous to all the samples, resulting in no amplification. Go to "set up" and, on the "assign targets to wells" tag, select the endogenous and select "none" in the reference. Then go to "analysis" and click on the "analyze" button. Magically, all your amplification curves will suddenly appear!
Hi there! I don't know if you solved this already, but just in case I write. I had recently this problem and came to this post to try to find a solution, I found it elsewhere in the end, so I am writing for all the future newcomers as well. The problem with this is that the machine is reading the ROX value on your endogenous control. If you didn't add ROX to your samples (like me), this makes the machine subtract the values of your endogenous to all the samples, resulting in no amplification. Go to "set up" and, on the "assign targets to wells" tag, select the endogenous and select "none" in the reference. Then go to "analysis" and click on the "analyze" button. Magically, all your amplification curves will suddenly appear!
Hi Nathanael,
Ur qpcr curves seem fine. As i see from your attachments, u have not set any threshold baseline.. set a threshold either automatically or manually. So that it lies at level of 0.4-0.5 of delta Rn because at that particular level, ur products have started to form (24-25cycles in X axis). Regarding, different heights of the three replicates in ur amplification curve, its simply because of manual error in pipetting that has caused formation of different amount of products in each well of triplicate. And so u get peaks at differnet level
Hi Nathanael,
Ur qpcr curves seem fine. As i see from your attachments, u have not set any threshold baseline.. set a threshold either automatically or manually. So that it lies at level of 0.4-0.5 of delta Rn because at that particular level, ur products have started to form (24-25cycles in X axis). Regarding, different heights of the three replicates in ur amplification curve, its simply because of manual error in pipetting that has caused formation of different amount of products in each well of triplicate. And so u get peaks at differnet level
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VOTE
This just looks like noise, combined with a rather high level of background fluorescence to me. It's worth remembering that logarithmic plots tend to amplify small variations around near-zero fluorescence. I suspect that if you did a linear plot, you would see nothing anomalous - though I prefer log plots for precisely this reason.
And a Happy New Year to you all!
This just looks like noise, combined with a rather high level of background fluorescence to me. It's worth remembering that logarithmic plots tend to amplify small variations around near-zero fluorescence. I suspect that if you did a linear plot, you would see nothing anomalous - though I prefer log plots for precisely this reason.
And a Happy New Year to you all!
More
VOTE
Hi Nathanael Quake and all others.
As the others have already told you, It doesn't seem to strange to call our attention ... But you should give us some more info in order to let us evaluate If it is strange or not.
For instance:
What is the ratio of primer to probe in your reaction?
Was it evaluated during your protocol optimization or did you use someone else1s protocol without changes?
What qPCR equipment are you using?
Could you send a pic of baselined and thresholded results or the experimental file to us?
What reagents and reaction protocol are you using?
How many cycles?
DNA, cDNA concentration?
With these data we may evaluate a little bit better and give you our impressions about your results. Ok?
If you need, just send a new comment here.
All the best for the new year to you all.
Hi Nathanael Quake and all others.
As the others have already told you, It doesn't seem to strange to call our attention ... But you should give us some more info in order to let us evaluate If it is strange or not.
For instance:
What is the ratio of primer to probe in your reaction?
Was it evaluated during your protocol optimization or did you use someone else1s protocol without changes?
What qPCR equipment are you using?
Could you send a pic of baselined and thresholded results or the experimental file to us?
What reagents and reaction protocol are you using?
How many cycles?
DNA, cDNA concentration?
With these data we may evaluate a little bit better and give you our impressions about your results. Ok?
If you need, just send a new comment here.
All the best for the new year to you all.
More
VOTE
Hi Subhasri Biswas , thanks for your answer. However, doesn't it seem strange that some curves do not follow a rectangular hyperbolic shape closely? I was wondering if (1) this is normal (2) what are the possible causes of these shapes.
This is another set of triplicates that I obtained recently. One of the curves does not follow a rectangular hyperbolic shape like the others.
Hi Subhasri Biswas , thanks for your answer. However, doesn't it seem strange that some curves do not follow a rectangular hyperbolic shape closely? I was wondering if (1) this is normal (2) what are the possible causes of these shapes.
This is another set of triplicates that I obtained recently. One of the curves does not follow a rectangular hyperbolic shape like the others.
More
VOTE
Hi there! I don't know if you solved this already, but just in case I write. I had recently this problem and came to this post to try to find a solution, I found it elsewhere in the end, so I am writing for all the future newcomers as well. The problem with this is that the machine is reading the ROX value on your endogenous control. If you didn't add ROX to your samples (like me), this makes the machine subtract the values of your endogenous to all the samples, resulting in no amplification. Go to "set up" and, on the "assign targets to wells" tag, select the endogenous and select "none" in the reference. Then go to "analysis" and click on the "analyze" button. Magically, all your amplification curves will suddenly appear!
Hi there! I don't know if you solved this already, but just in case I write. I had recently this problem and came to this post to try to find a solution, I found it elsewhere in the end, so I am writing for all the future newcomers as well. The problem with this is that the machine is reading the ROX value on your endogenous control. If you didn't add ROX to your samples (like me), this makes the machine subtract the values of your endogenous to all the samples, resulting in no amplification. Go to "set up" and, on the "assign targets to wells" tag, select the endogenous and select "none" in the reference. Then go to "analysis" and click on the "analyze" button. Magically, all your amplification curves will suddenly appear!
More
VOTE
It looks pretty normal(red curves), which is your the value for the baseline, and the values for the Cts (apply a threshold).
How much template did you put in your reaction?
It looks pretty normal(red curves), which is your the value for the baseline, and the values for the Cts (apply a threshold).
How much template did you put in your reaction?
More
VOTE