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  • NaOH/clustering

    Hi~we are new users to the HiSeq and MiSeq. We are getting VERY low cluster densities after quantifying by BioA, Qubit, and qPCR. We have been told to check the pH of the NaOH we use to dilute our samples in. Has anyone had problems with this?

  • #2
    MiSeq platform- We have not had issues using NaOH, side note though, we use the illumina brand 2 N NaOH that comes with the PhiX control. Also, if you dont already, use the PhiX control with your library it will help with any troubleshooting.


    • #3
      We've heard of reports of problems with NaOH shipped on dry ice. If the vial isn't completely sealed the NaHO reacts with the CO2 and the pH goes down. This can result in reduced efficiency and a shift in GC bias. I know some sequencing centres are making up their own NaOH stocks rather than using the stuff which ships with the kits. I have to say it's not a problem we've seen ourselves but if you are having problems then it's an easy and safe thing to change.


      • #4
        From manual of TruSeq DNA sample preparation V2, with step of DNA denaturing, mention that DON'T apply NaOH over 800 uM in the final concentration since afterwards the cluster generation will be impaired if the NaOH conc is above 800 uM.