Based on the info above, 10 - 6.5 (5 uL mastermix, 0.5 uL x 3 Dye Mix) is actually what you can use for sample input which is 4.5 uL...so you don't have to use 1.0 uL of water. They gave you the reaction for 1x, all you have to do is multiply by the reactions that you intend to do plus 10% for dead volume or pipetting inaccuracies as per the usual. So if you wanted to do 96 reactions, you would typically add around 10 more reactions to a total of 106 reactions. So that would mean based on your reactions above:

Master mix = 5 ul * 106 = 530

Dye Assay mix (Target 1) = 0.5 ul x 106 = 53

Dye Assay mix (Target 2) = 0.5 ul x 106 = 53

Dye Assay mix (Target 3) = 0.5 ul x 106 = 53

Nuclease free H2O = 1.0 ul x 106 =106 (if you decide to use...you don't have to)

Just change the 106 to 360 for your reaction and that's what you need to do 360 reaction (don't forget the extra 10%). Probably do this in a 1.5 mL PCR tube to set it up and vortex/spin down. If you want to put more DNA/RNA into the reaction, you could probably omit the water part. But if you are happy putting 3.5 uL of input instead of 4.5 uL, that could work too.

Can Kiessling Thank you for the prompt response.

The kit states that 0.5 ul of each labelled assay needs to be added, but the custom 20x Triplex assay kit already contains all three labels. The question is do I add 0.5 ul of the 20x triplex assay to the 7 ul master mix reaction, or do I add 0.5 ul for each label (as you suggest), thus 1.5 ul in total of the 20x triplex reaction to the 7 ul master mix reaction? Kindly find the assay names below.

Triplex 1. Rn01775763_g1 - Gapdh 2. Rn01463997_m1 - Crhr1 3. Rn00561409_s1 - Htr1a

Thank you

What is your final volume for the reaction? It is almost usually 5, 10 or 20 uL. From what you wrote, to me it seemd like 10 uL. If it is 10 uL, then Dye Assay mix being 20x makes sense because 10/20 = 0.5 uL which is what you stated as input for 1x reaction.