i had run the std by using taqman probes by using same dilutions made from 100% methylated sample. but the pcr effiency is poor. so should i have to make the dilutions from same sample or from other sample and should i have to change reagents too? what does y-intercept and error value indicate in pcr.
I have attached the files of std curve check it out and let me know what should i have to do? i'm a student and i joined in research 2 months before
To be a valid experiment your CC should have atleast 5 calibration points. While diluting the initial std to further down, taking care on pipetting is a known crucial part.
Make sure your reagents including standards are intact in quality for your experiment.
Take care while diluting standard.
Good luck!
I strongly suggest to discuss these things with your supervisor.
There are plenty of books available about real-time PCR, you will surely find many of them in your library. It is helpful to have a look at such books. Actually your supervisor should have already proposed some literature about this technique.
Additionally I suggest that you learn more about statistics in general and linear regression in particular.
But to give you some concrete help here:
Lakshmi is correct. You need more different dilution steps (it is not required to mesure the same dilution several times). Ct values greater 35 are likely unreliable. The upper diagram shows a complete mess. And regarding the intercept in your case. you fitted an exponential model Ct = m*log(Quantity) + b. The intercept is b, and this is the Ct value for which log(Quantitiy)=0, that is, where Quantity=1. What you mean with "error value" is not clear to me. Any estimate has its error. In a regression analysis there can be a MSS, SE(Y), SE(m), SE(b), and there are different ways to express an error of an estimate (e.g. as variance, standard deviation, standard error, confidence interval).
okay i get it. But my PI want me to solve this issue by myself.
Today when i spoke to her about this she has asked me about what to change in this assay whether it is reagents or dilutions and i said after checking several articles and also by life technologies in youtube. i told that i have to make new dilutions then she said to me that why changing the dilutions and i said that due to dilution error the std had shown error. so again she asked me about the the 2 assays which i posted for this both assays should i have to make dilutions or should i have to change the reagents like primers and probes. Actually what i got in test pcr all assays were fine but when coming to std curve all are showing problems. i don't know what to do now could u help me out plzz
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