[FONT=Arial]Hi everyone,
Do anyone know why is possible to get more than 100% efficiency in Real-time PCR? I don't have neither primer-dimer or non-specific products and it doesn't happen always. What can I do to optimize this?
Thanks a lot.
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#2
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Posted 27 January 2005 - 03:14 AM
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Do anyone know why is possible to get more than 100% efficiency in Real-time PCR?
Why should this be possible?
#3
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Posted 27 January 2005 - 04:15 AM
nabla, on Jan 27 2005, 04:14 AM, said:
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Do anyone know why is possible to get more than 100% efficiency in Real-time PCR?
Why should this be possible?
Sorry, maybe i didn't explain it well (english is not my lenguage). I am upset because I use to get efficiency of 120% and more so the amount of DNA is doubled in less than a cycle. How does it is possible?
Thanks again
#4
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Posted 27 January 2005 - 05:47 AM
How do you calculate the 120%?
I only know ratios of </=2,0. Practically the DNA can only be dublicated in case of an efficiency of 100%.
Values above 100% are incorrect approximation caused by the calcuation methode, as far as I know!
When you have optimized Mg-concentration, the cycle program, amount of primers and probes then you can not do so much more. There are always reactions (pcr) that are difficult or slow, so you can't really do more than optimize the standard parameters. Everey template is subject to a individual kinetic furthermore steric problems are often difficult to overcome.
I only know ratios of </=2,0. Practically the DNA can only be dublicated in case of an efficiency of 100%.
Values above 100% are incorrect approximation caused by the calcuation methode, as far as I know!
Quote
I don't have neither primer-dimer or non-specific products and it doesn't happen always. What can I do to optimize this?
When you have optimized Mg-concentration, the cycle program, amount of primers and probes then you can not do so much more. There are always reactions (pcr) that are difficult or slow, so you can't really do more than optimize the standard parameters. Everey template is subject to a individual kinetic furthermore steric problems are often difficult to overcome.
