2 of my primer sets didn't reach plateau aft 40 cycles, while the rest did at ~30 stg. Can i proceed since we don't need the plateau phase for any calculation? Or should i work it out so that it can reach plateau aft the reaction? How can i work it out? Adjusting the primer concentration?
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Is it acceptable that qpcr reaction doesn't reach plateau?
Started by hianghao, Apr 19 2011 03:49 AM
3 replies to this topic
#2
Trof
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Posted 19 April 2011 - 05:03 AM
I would say it depends on how is your Ct calculated. If you're using fit-points method, (setting treshold) I'm pretty sure it doesn't affect that. If you have for example LightCycler 480 software and use second-derivative method I'm not sure. But as I recall it didn't have problems either, I had samples like that occasionaly.
Anyway, the important part of the curve is the exponential, so in general this should not be a problem.
Anyway, the important part of the curve is the exponential, so in general this should not be a problem.
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#3
Prep!
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Posted 19 April 2011 - 07:52 PM
i second trof.. in fact i know people who to save time have not run the q-pcr till the plateau phase on purpose!!! 
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#4
hianghao
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Posted 20 April 2011 - 03:08 AM
Trof, on 19 April 2011 - 05:03 AM, said:
I would say it depends on how is your Ct calculated. If you're using fit-points method, (setting treshold) I'm pretty sure it doesn't affect that. If you have for example LightCycler 480 software and use second-derivative method I'm not sure. But as I recall it didn't have problems either, I had samples like that occasionaly.
Anyway, the important part of the curve is the exponential, so in general this should not be a problem.
Anyway, the important part of the curve is the exponential, so in general this should not be a problem.
Prep!, on 19 April 2011 - 07:52 PM, said:
i second trof.. in fact i know people who to save time have not run the q-pcr till the plateau phase on purpose!!!
Thanks for the information!
