Abnormal plot of real time one-step RT-PCR, how to solve! - (Feb/22/2012 )
i have a problem with real time one-step RT-PCR.
My sample is total RNA and i detect one target in that with a specific primer pair & taqman probe (FAM).
i ran the experiment with 2 types of sample:
- Target sample: include template, primer, probe, buffer for one-step PCR (Curves are red lines)
- Negative- control: (green lines)
+ two higher green lines : only primer, probe, no template & buffer
+ Two lower green lines: only water & buffer, no primer & probe
As you see in the picture attached in this mail, abnormal curves are rough, zig-zag, (rise up slightly in negative controls)...not smooth.
Everything i used in this assay is new with many times of repeat but the results get back are like that.
So everyone please help me any advice to solve it.
Thanks you very much.
Those look great to me. Some cyclers smooth the readings, so you may be used to "better" looking results. But your controls are negative and your samples are positive and pretty consistent. What more do you want?