Ok, well firstly you can see that at 100mM each, needing a final concentration of 25mM, you're going to be adding equal vols of each, with no spare volume whatsoever.

25ul CTP, 25ul GTP, 25ul ATP, 25ul UTP = 100ul of stock at 25mM each.

This means that unless your biotinylated CTP is also at 100mM (which it isn't), you cannot replace any of the CTP with bio-CTP and keep vols and concentrations constant.

But that's ok, because making that stock is just _convenient_, not required. And the kit clearly allows for 50ul of spare volume in the "template plus water" part.

So think of it as volumes instead of concentrations.

For the '20ul of rNTPs', you're essentially adding

5ul GTP, 5ul ATP, 5ul UTP and 5ul CTP.

And you instead want to add 5ul GTP, 5ul ATP, 5ul UTP and 4.5ul CTP.

You then want to add surplus bio-CTP to make the total CTP correct, but your stock is only 10mM not 100mM, so you need to add 5ul (not 0.5ul)

You then just reduce the "water plus template" vol (currently 50ul) to 45.5ul, to account for the fact you've added an extra 4.5ul of liquid already.

Or, to streamline things for future, make a stock with 25ul each of GTP, ATP, UTP and 22.5ul of CTP, plus 25ul of bio-CTP. Use 24.5ul of this per reaction (rather than 20ul)