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Smart Cycler II software and Absolute quantification help

Efficiency and slop

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#1 Abd.Obeidat

Abd.Obeidat

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Posted 09 June 2013 - 05:42 AM

Dear friends and colleagues,

I am having a problem regarding quantification of HBV samples using the smart cycler II. The method is absolute quantification via standard curves.

According to my knowledge, If we have an unknown sample with quantity lower than the lowest standard used in the experiment, the software will quantify it using an equation and will give me the quantity of that unknown sample. the lowest standard concentration is 2500 IU/ml.

what I am facing, that I got an amplification for some sample, with ct value usually 38-41 (the reaction protocol up to 45 cycles), but the software does not give me a value for those sample, and on the standard curve it is clearly out of the curve. And according to the method we are using it is sensitive to detect down to 20 IU/ml.

I want to know, what shall I consider these samples? they have an amplification curve but very late, shall I quantify them manually? what to do? this lead me into another question, where I can find the efficiency and the slope of the standard curve in Smart Cycler Software.



#2 Shefali Desai

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Posted 09 June 2013 - 10:59 PM

Hi!
If your cT value is 38-41 you should concider samples as negative.Have you checked the sensitivity in your lab? Becauseyou need to validate any kit in your lab and find out what is highest and lowest values you can detact in your lab. You also need to need to run stds which would cover your expacted values. lowest standard concentration is 2500 IU/ml is too high.




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