Urgent - MiSeq 12.5pM PhiX concentration

[seqgirl123]

If I use the MiSeq v3 kits, according to the denature & dilute guide (attached), I dilute both my PhiX and library concentration to the same concentration, and spike in the desired Phix percentage. For example, if I want to load 15pM at 25% PhiX, I denature and dilute both Phix and library to 15pM, then 150uL of 15pM PhiX + 450uL of 15pM library.

If I use MiSeq v2 kits, the guide says to dilute PhiX to 12.5pM. If I wanted to load the same 15pM at 25% PhiX, what is the calculation? Am I having PhiX at 12.5pM and the library at 15pM dilution? How to do this for v2 kits?

[Jessica_L]

if you have 1000ul of library at 15pM, you should have 15000pmol of material. 25% of that should be 3750pmol (that'll be your PhiX target). There are three ways to proceed:
From the 20pM denatured stock, use 187.5ul. (187.5 * 20 =3750)
From the 15pM denatured/diluted PhiX, use 250ul (250 * 15 = 3750)
From 12.5pM denatured/diluted PhiX, use 300ul. (300 * 12.5 = 3750)

That should get you close to the desired spike-in, I think.