problems with dilutions: sensitivity - (Nov/07/2006 )
Hello again guys: 
I have a new problem today. I am working with a honeybee virus and to detect it I do SYBR GREEN Real Time. My problem is that I have a positive sample and I do dilutions but then, on the amplification plots, the dilutions don't seem to be dilutions, their Ct is the same or very similar to the positve sample not diluted. At the beggining I thought that I was making lot of errors making the dilutions but after repeating them hundred times, and by different people, I started thinking that it was't the main problem. Do you think the primers aren't good enough to detect the differences between the dilutions??
How did you prepare your dilution? How much primer did you used?
Normally, people prepare 10 fold serial dilution.
More likely one of your reagents has contamination. Do you run a negative control?
You should run a negative control on your dilution reagent as well, by loading a negative sample and performing a serial dilution series on it.
Hello:
I do ten fold dilutions and use 400nM of each primer. I optimized the quantity of primers doing a matrix of concentrations and I always do a negative control so I know there is no contamination. I can't think about other problems and don't know what to do.
dear debokuki,
Could you please attach your amplification curve along with lable and discription?
Thank you very much
Could you please attach your amplification curve along with lable and discription?
Thank you very much
Hello!
Here I attach you two of my assays. Both are the same sample diluted in ten fold dilutions. Also I attach the dissociation curve wich is a bit strange because the dilutions have a different curve than the not diluted sample.
Please, take a look.
Thanks!
Dear debokuki,
Sorry for the late reply 
From where your positive control derived from? Is tissue culture (PFU) or PCR product?
I don't think is you primer problem, because if your primers aren't good enough, you should have low efficiency, and the effect should be applied evenly on every dilution.
Ct value is determine by initial template present in solution, so I think is still you dilution problem.
Hello Debokuki
sorry for re-opening an old topic...fact is, i have much the same trouble - diluted samples dont always give a difference in Ct, or the difference much smaller than expected...so i am dying to know, have you solved this problem? and how...?!
wish you best of luck with your project.
Hi everybody! Sorry again for re-re-opening this topic 
... I feel better since Im not the only one having these dilution problems.
My standard curve with genomic DNA works fine, but lately my ten fold dilutions of cDNA are giving the same Ct... I've tried with a new synthetized cDNA, but this time the Ct was too much larger between ten fold dilutions than expected!! This way it is impossible to obtain consistent results 
Please!! anyone knows why could this be happening? Maybe I have a nuclease or some other contaminant in my cDNA?? Or is it too saturated or too diluted to show the expected difference in the Cts?
Please please heeelp... I cant continue wasting more time on this! 
Thanks a lot! kisses
Hi leahf and kukita,
Just to remind you yugs that to be accurate, you need at lest 2ul of your PCR product/cDNA to strat with......
and give a good mix to each dilution before you trasfer to next tube (somebody even change tip for every dilution).
Also check you primer efficiency, if you primer forming primer dimmer or hairpin loop, it will affect to your Ct value.
Best regards