Primer calculation for qPCR - final reaction - (Jun/10/2015 )

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 :)

Mad Researcher on Wed Jun 10 14:53:39 2015 said:

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.