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The pool via standard size estimation returned a mean of 42.66 nM. This should be adjusted for the difference between the standards' fragment sizes and the expected product size (452 vs 481). 42.66x(452/481) = 40.087 or ~40 nM
Sequencing Test:
Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.
1000/Results = ul of Pool to Add
1000/40 = 25uL of Pool to Add
1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add
1000- 25 = 975 uL of 10mM Tris 8.5
Dilute 1 nM full pool to loading concentration of 50 pM:
Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX
Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.
Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100
Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.