Hello all.
I have been working with a sequence that is a 571 bp clone, and I have a primer pair that gives a 150 bp amplicon. When I ran the system in real time for a calibration curve I got some results I could not explain. First of all, the highest concentration sample (1000 ng/ul) did not give a signal at all. The next dilution in the series (100) gave a signal of around 30, and the serial dilutions where around 1 or 2 increments (needless to say the eff. value is very high and basically unusable, around 300%). Raising primer conc., doubling SYBR green mix amount and lowering annealing temp had no real effect. Adding DMSO (5%) had a good effect on sample variability, but not on the Ct values. When I ran the reactions on a gel I got a very good product for the first 4 dilution, even for the sample that did not generate any Ct to speak of. Does anyone have an explanation for this lack of correlation between getting the product and not getting a Ct value? If you encountered something like this before, what steps did you take to improve the real time reaction?
Thank you
Ori Elad