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realtime PCR interpretation-peak found in negative control but no Ct value


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#1 pop09

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Posted 12 November 2009 - 11:18 AM

Hi all, I have posted earlier some questions about interpreting melting curves. I've been doing optimization with my protocol. I started off by doing a gradient temp in the realtime PCR machine. I did not get any Ct value (N/A) for my negative control, however, I got Tms which were the same as my positive samples. When I looked at the melting curve, the lines for the control were either straight or had a little bump at the said Tm.

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.

#2 ivanbio

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Posted 12 November 2009 - 12:07 PM

Hi pop09,

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.

Ivan
Carlsbad, CA

#3 pop09

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Posted 12 November 2009 - 03:54 PM

Ivan, thank you. Regarding my gradient temperature run, I observed that as I increase the temperature, the Ct value increases as well. previously, i did a regular PCR and found that my target DNA is amplified well at 58C, and becomes a lighter band by 62C and no more band at 66C. So I decided to do a gradient temp in real time PCR using 58, 60 and 62C. As the temperature increased, the Ct value also increased slightly. How would I use this information to decide which temperature to use in my final runs? Also, I tried 2x sybr green mix and 1x sybr green mix stocks. The Ct values behaved the same way with temperature but I am getting higher absolute values with 1x than with 2x.

How should I proceed from here based on Sybr green mix stock concentration and temperature?

#4 Mighty Mouse

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Posted 12 November 2009 - 04:50 PM

Have you examined the efficiency of your primer at the various temperatures? This may answer your question to some extent...the temperature in which you have the best efficiency seems like it would be the best temperature to use. I know when I run my primer matrix I typically go for the primer concentration that gives me the lowest Ct value with the best looking melt curve. So ceteris paribus (all things being equal) go with the conditions that give you the lowest Ct value, thereby maximizing your sensitivity.
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#5 pop09

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Posted 12 November 2009 - 07:28 PM

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?

#6 Prep!

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Posted 12 November 2009 - 07:37 PM

hey pop nice to see u working on ur pcr conditions.. ya i too think that u shud also vary your primer conditions... and as mm said the lowest Ct value shud be considered the best along with a good melt curve... i think u are almost there and by this final step of primer optimization u shud be thru.. best luck!!
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#7 Mighty Mouse

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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.
We are all artists...painting with experience on the canvas of life




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