At 1 day, 10hrs into the run (~12hr after wash #1), performed a second wash on the MinION flow cell.
First, needed to get a library of 200fmol. I previously diluted a portion of G1L4 to 100fmol/12uL, in a total of 36uL. I used 12uL of this for the first load, so I now have 24uL of diluted library. I also still have some undiluted Library 4. I need to add some undiluted Library 4 to my dilution to raise the concentration to 200fmol/12uL (since I observed higher pore occupancy with more input DNA).
Calculate how many uL of undiluted Library 4 to add:
A = concentration of Library 4 = 36.6 ng/uL
B = concentration of dilution = 100fmol/12uL = 43.1ng/12uL = 3.58 ng/uL
v = volume of dilution = 24uL
x = volume of Library 4
\(\frac{total\ DNA}{total\ volume} = \frac{(Bv+Ax)}{v+x} = 2B \ \ \ \Rightarrow \ \ \ x(A-2B)=Bv \ \ \ \Rightarrow \ \ \ x=\frac{Bv}{A-2B}\)
\(x=\frac{Bv}{A-2B} = \frac{3.58\frac{ng}{uL}*24uL}{36.6\frac{ng}{uL}-2*3.58\frac{ng}{uL}} = \frac{85.92ng}{29.44\frac{ng}{uL}} = 2.92uL\)
Added 2.92uL of undiluted G1L4 to the MinION dilution to bring the concentration up to 200fmol/12uL. this is twice the recommended concentration for MinION loading.
I then used this newdilution to wash and reload the Group 1 Library 4 flow cell.
541 pores (129 reserved, 412 available) before wash, 693 pores (255 reserved, 438 available)