10ul of 700pM library. How to Denature and cluster?

[yoyoming1001]

Hi, We have 10ul of a low quantity library (700pM) that we are trying to denature and load onto the flow cell at 10pM.

So far, the best that I can come up with while still keeping the NaOH concentration low enough is 9.5 ul of the sample + 0.5 ul of 2N NaOH and load all 10ul in 990ul of HT1, giving a final pM concentration of 6.65. Unfortunately this won't give us the cluster density that we would like. I could adjust the HT1 volume but that would increase the NaOH and probably inhibit clustering somewhat, if not all together. Could I double the incubation time and decrease the NaOH concentration by half? I'm not sure if the denaturation would work at half the normal concentration. Does anyone have another solution? Any suggestions would be really appreciated.

[HESmith]

Dilute libraries can be neutralized with HCl after denaturation, then adjusted to the appropriate final concentration with HT1. For 700 pM library, use 2 ul in standard 20 ul denaturation reaction. After placing on ice, add 2 ul 1M HCl, then 118 ul HT1 for 10 pM final concentration.

[yoyoming1001]

Thanks so much HESmith. It worked! The only difference was for that one sample the cluster number was a bit higher than the other samples. I wonder if that is expected since the neutralized NaOH won't inhibit template binding in any way.

Anyway, Thanks again.