Unfreeze the Pool, PhiX, Flowcell, HT1 and Reagent Cartridge (Illumina)
HT1 and Reagent Cartridge can thawed in water to save some time.
Take 2 empty 1,5ml Tubes and dilute NaOH(dilute to 0.2N) and Tris (dilute to 200 nM) with UltraPure Water (total 100µl dilution).
Take 2 empty 1,5 ml Tubes for Denaturation and final Dilution.
Denaturation: mix 5µl the ~ 4nM Pool and 5 µl freshly diluted 0.2N NaOH
Vortex briefly and then centrifuge at 280x g for 1 minute
Incubate at room temperature for 5 minutes
Add 5 µl Tris-HCl, pH 7
Vortex briefly and then centrifuge at 280x g for 1 minute
Dilute denatured Pool to 20 pM with prechilled HT1. How much HT1 you need depends on the pool conc. (see in Pooling sheet)
Vortex briefly and then centrifuge at 280x g for 1 minute
Place the 20 pM pools on ice until you are ready to proceed to final dilution