RT-PCR - problems with TaqMan PCR (Sep/17/2009 )
I have been running TaqMan assays using Roche LightCycler 1.5 software version 4. I have gotten some strange results. I clearly had some positive results with nice curves and Cp values of around 19-21 and fluorescence around 7-9 (not sure of the parameters in measuring fluorescence as i do not have my data directly in front of me)
My no template control had a positive result. which was strange considering I used water. Some of the other controls that should have been negative had Cp values. They were high though. Also the fluorescence was around 0.31 to 0.5 for those that should be negative.
my positive control did not work. I think the cDNA degraded. I have had previous success using the cDNA from the same sample where it had a Cp value of around 21.
My questions are does anyone know how to determine positive or negative results using Cp values. I have read some research articles where they say if the results are <35 or less than <38 then its negative. How do they come up with these values and justify it for publication?
The stuff I am working on I am hoping to publish but I cannot have shady data.
When I say <35 or <38 I am referring to Cp values
Does anyone know if you can upload positive control data from a previous experiment using the LightCycler software?
Can you show us your curves and mark where is yes/no template ?
The first thing comes to mind is that you have a pcr-product contamination.