I need an urgent help with q-pcr amplification plots - (Oct/28/2010 )
Hi all again,
I think I found the reason of the weird amplification plot 
I considered the technical note #5 in the Bio-rad's HRM-tech note paper which is suggested by Nigntingale. There was an efficiency problem in my experiment because, the fluorescence remained at low levels and efficiency calculated from standard curve was low also. In the tech-note #5 it is proposed that jagged-plots could be the result of a poor amplification. Therefore, I decided to decrease the annealing temperature. First, I decreased temperature from 62 to 60 and got smoother plots. Then I decreased 2 degrees more and set the annealing temperature to 58 and I got smoother and smoother plots. Also fluorescence increased up to 100 from 20s and efficiency increased to 100%. I understood that the problem was annealing temperature. I will continue with this annealing temperature, I hope there will not be a problem in the melt curve for the oher samples those were not included during optimization.
I want to thank Nightingale very much for the effective references and the others for all the contributions. I hope this discussion helps all of you.
genom38 on Sun Nov 14 21:27:30 2010 said:
Hi all again,
I think I found the reason of the weird amplification plot
I considered the technical note #5 in the Bio-rad's HRM-tech note paper which is suggested by Nigntingale. There was an efficiency problem in my experiment because, the fluorescence remained at low levels and efficiency calculated from standard curve was low also. In the tech-note #5 it is proposed that jagged-plots could be the result of a poor amplification. Therefore, I decided to decrease the annealing temperature. First, I decreased temperature from 62 to 60 and got smoother plots. Then I decreased 2 degrees more and set the annealing temperature to 58 and I got smoother and smoother plots. Also fluorescence increased up to 100 from 20s and efficiency increased to 100%. I understood that the problem was annealing temperature. I will continue with this annealing temperature, I hope there will not be a problem in the melt curve for the oher samples those were not included during optimization. I want to thank Nightingale very much for the effective references and the others for all the contributions. I hope this discussion helps all of you.
am overwhelmed by your nice message
thank you ...
second to that : am really glad that by referring to my suggested, your matter has seen light out there
Wishing You All The Best