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Quantification and Internal Standard

Quantification and Internal Standard - Real-Time PCR and Quantitative PCR Forum

Quantification and Internal Standard - Real-Time PCR and Quantitative PCR Forum


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Old 07-17-2010, 02:20 PM
Pipette Filler
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Default Quantification and Internal Standard



Hey all,

i try to establish a sybr q pcr wich detects two different genes. ive already cloned my genes and a standard dilution was done. Followed by optimization of MgCl2 and pimer concentration.
Due to the reason that i try to quantify aqueous bacteria via this two genes i designed and IC which runs with the same pcr conditions.
But my question is:
I calculate via the cp of my standard dilution my concentration of desired genes. Ok, but if there is an influence like inhibition due to the seawater i can detect this via my IC. So how would you calculate the inhibition of the desired genes in the sample in relation to the IC.
For example:
Standard 1E2 concentration with Cp 24; sample 1 5E1 concentration with Cp 27.
IC in a.d. Cp 24; IC in environmetal sample Cp 30.
This means a delay of 6 Cycles of the IC.
So there is an influence visible but how should i implement this delay in final sample concentration calculation?

Thanks for your help
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