I think there is a difference between these 2 dillution methods and therefore it might affects the reaction (A+B=AB). Could you please give me some clear explainations on this:

I have:

Stock A: 10 M

Stock B: 1M

I would like to make a final solution of 120 uL of 0.5M B and 2.5 M A

Method 1: Make 5M A solution by dillution, then take 60uL of 5M A and 1M B, mixing, done

Method 2: Take 30 uL of 10M A, then mix with 60uL B, finally add 30 ul DW.

(note: 30 ul of 10M A and 30 uL DW if mixed could make 5M A, but it is separated here by mixing with 60 uL B prior to DW)

Do you think Method 1 and 2 would result in different reaction product outcomes, for example: The Quenching effect between Fluorophore and Quencher.

Hope to have your opinions. Thank you ~