strange QPCR amplificaton plot - (Sep/30/2005 )
How are you all?Today I encounter a strange amplification plot of my RT-QPCR. Does anyone know why the two most middle amplification plots have such strange pattern? They just have the shape of which I would love to laying on it and read my favorite romance!
Any reply will highly be appreciated.
i would say you have a pretty low starting template
the curve is nice and smooth, but taking a loooonnnnngggg time to hit good amplification
what is your efficiency for that product?
First of all, please tell us what system are you using?
SYBR Green / TaqMan / M. Beacon / FRET / LUX?
Have you included your negative control in these plot? Which one?
What product arre you using? One step RT-PCR or two steps?
Have you successfully rub this assay before?
Dear aimikins and Hadrian,
Thankyou for your kind response
Here are the details of my samples:
My system is ABI75000 and I use SybrGreen. I use cDNA as my template. The reactions had the same melting temp.
I have run this assay before and it was ok. The only thing what I suspect now is my primers. Because it also gave the slowly raising plateau in the blanco. Only it missed the most right slope. Signal from my samples could only be detected when its signal is greater the the backgrond. Although not all my amplifications have this same problem when using this primerpair, I think it is not reliable any more.
Thankyou again for the inputs of you two. Have a nice day, friend!