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Old 05-07-2019, 11:38 AM   #6
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Location: Washington, D.C. metro area

Join Date: Feb 2010
Posts: 118

The NaOH concentrations are about what they should be for each individual step of the process. The only thing I haven't seen before is starting with a 10nM library-- the standard protocols I've seen all recommend starting with 4nM. I'm not sure that makes a difference, but it might be worth trying. Starting with 4 can still get you to 20pM if you load at that concentration.

If you need to get the library to a higher concentration, you can follow the procedure that ArcherDx uses in their protocols, which generate a 40pM denatured library:
10uL of 4nM library + 10uL of 0.2N NaOH
Incubate 5 minutes at RT, then add 10uL of 200mM Tris pH 7
+970uL of HT1
The final concentration is a 40pM library and ~2mM NaOH. Since the library is more concentrated at this step, you use less of it, and dilute to a final loading volume of 1000uL.

I've used both the standard Illumina protocol and this one, and had no problems with the cluster generation part of things, but again, we start with 4nM.

Good luck!
Jessica_L is offline   Reply With Quote