Finding an answer as to why I have Tm but no Ct - (Jun/30/2014 )
Hello!
I am currently performing a qPCR on a LightCycler 480 in the way of melt curve analysis. I get a peak at the appropriate temperature but no there is no amplification on the Ct aspect. Can this be regarded as a sample that has the virus present? Why does it do this? Does it need to be re-ran/re-extracted?
Thank you in advance for any assistance.
Hmmm....have you run a gel with the products, and were there any bands (if yes, which size) ?
Most likely you don't have a real peak.
If you are running SYBR or other intercalator dye assay, it's not possible to have a (high) melting peak without amplification.
You either have an amplification curve that is too low and late to be seen (and too late for the Ct to be calculated) and/or you have your melting peak just very low, but alone it looks higher.
You need to look at the amplification curve alone in the graph (if there is any amplification at all, in the later cycles) and you need to compare the Tm peak with the other (possitive) samples you have.
Ideally make a screenshot of both.
Tabaluga on Tue Jul 1 08:07:40 2014 said:
Hmmm....have you run a gel with the products, and were there any bands (if yes, which size) ?
Have not. Do not have the proper equipment to perform one.
Trof on Tue Jul 1 12:38:54 2014 said:
Most likely you don't have a real peak.
If you are running SYBR or other intercalator dye assay, it's not possible to have a (high) melting peak without amplification.
You either have an amplification curve that is too low and late to be seen (and too late for the Ct to be calculated) and/or you have your melting peak just very low, but alone it looks higher.
You need to look at the amplification curve alone in the graph (if there is any amplification at all, in the later cycles) and you need to compare the Tm peak with the other (possitive) samples you have.
Ideally make a screenshot of both.
If I compare the Tm peak of a positive sample to an uncertain sample how could I distinguish how far I can go down the Y-axis before regarding it as negative? Thanks for the help! :)
It's hard to say. I don't think you can set any definitive cut-off. But negative sample usually seem flat, when you select both negative and positive ones for graph display.
I have LC480, so I may be able to tell you from screenshots.
Trof on Wed Jul 2 09:49:25 2014 said:
It's hard to say. I don't think you can set any definitive cut-off. But negative sample usually seem flat, when you select both negative and positive ones for graph display.
I have LC480, so I may be able to tell you from screenshots.
I can show you a couple tomorrow! :)
Have you set the PCR protocol correctly? For melting curve PCR, you should set READING STEP two times: during PCR and during melting curve analysis if you want to see both Ct and Tm.
I did face this problem one time, b/c I forgot to set READING STEP during PCR, so I could not see the Ct. ^-^