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Primer calculation for qPCR - final reaction

Primer qPCR final reaction

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#1 Mad Researcher

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Posted 10 June 2015 - 06:53 AM

Hi,

 

I am not sure if i am doing it right.

 

I have 100 uM stock conc. of primers. I have made working solution of 10 uM conc. I further made working solution of 0.3 uM of my primers as i need to use 0.3 uM in my qPCR reaction.

 

I am taking 1 uL of the 0.3 uM primer but i guess i am taking way too much. How do i calculate the vol. of primer that i have to take for my final PCR reaction?

 

I appreciate your help in understanding this.

 

Thanks :)


Cheers,

Mad Researcher

#2 r.rosati

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Posted 10 June 2015 - 11:27 AM

I further made working solution of 0.3 uM of my primers as i need to use 0.3 uM in my qPCR reaction.


...please be sensible, you can't make a 0.3 uM stock if your final concentration must be 0.3 uM and it gets mixed with other things, right? Which concentration is it going to be if you take 1 ul and dilute it further in the PCR mix?
You need to take the 10 uM stock, and add it to the PCR mix so that it has a final concentration of 0.3 uM.
This is a simple dilution. Come on, you can do it: it's one of the very basic skills of the trade, and involves math skills which are well below second grade.

Edited by r.rosati, 10 June 2015 - 12:37 PM.






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