Failed

Failed to obtain result of qRT-PCR. This is the first time that I did Real Time PCR. I was expecting enhanced expression of tatA, tatC and per - genes in presence of iron among WT and fur-mutant Listeria monocytogenes. 16S rRNA gene was used as an endogenous control. The most surprising thing is that none of the 3 x 3 x 2 x 4 = 72 reactions was observed with characteristic sigmoid curve; rather all the lines were in log phase only without labeling off. So, the instrument could not calculate Ct value for any of the reaction triplicates. My instructor mentioned that I did wrong somewhere during reaction preparation. However, my boss suspects on quantification of mRNAs and/or cDNAs. Only the thing that I know is I failed the experiment at the first time. I was so scared to report the case to my professor. However, he simply said me to repeat the experiment. I have started right from the beginning - growing bacteria.