realtime PCR interpretation-peak found in negative control but no Ct value
Posted 12 November 2009 - 11:18 AM
Can someone please explain how I can have a Tm value for mynegative control, but not have a Ct value for the same sample?
thankyou so much.
Posted 12 November 2009 - 12:07 PM
The simple answer to your questions is as follows: the threshold line that the instrument uses to extract Ct values is automatically set at a level that is higher than background noise. If the signal generated by your negative control is lower than this threshold line, then you will get no Ct value, even though there is enough signal present to show up as a little bump in your melt analysis. Another way to say this is: the sensitivity of the software in identifying the Tm value is higher than the sensitivity of the same software is extracting a Ct value. Of course you can always manually lower the threshold line and you should be able to get a Ct.
Hope this helps.
Posted 12 November 2009 - 03:54 PM
How should I proceed from here based on Sybr green mix stock concentration and temperature?
Posted 12 November 2009 - 04:50 PM
Posted 12 November 2009 - 07:28 PM
Posted 12 November 2009 - 07:37 PM
Posted 13 November 2009 - 08:03 AM
Thank you Mighty Mouse for your reply. I have been working with a final concentration of 0.1 uM of each primer. Operationally, what do you mean by efficiency of primer concentration? Do I run different primer concentrations with my temp gradient?
Perhaps I wasn't clear with what I meant. What I mean is, test your primer efficiency using the best primer concentration (in your case it sounds like your using 0.1 uM for everything, so if this works I would just keep it the same) at your various temperatures to figure out which condition gives you the best efficiency (i.e, 100%). Then use that for your experiments.