I have made a DNA library for miseq using truseq pcr-free dna HT kit (550bp insert). The problem is that at the end of library preparation and pooling, I need at least 2nM of library for denaturation. But I have only 1nM left in 10 ul. So I can not concentrate it more. (quantified by Kapa universal library quantificatiun kit).
My question is that can I modify the protocol by denaturing in following condition
2 μl of 0.5 N NaOH + 8 μl 1 nM DNA library
instead of recommended
5 μl of 0.2 N NaOH + 5 μl 1 nM DNA library
Then I would have 800pM after denaturation and with a final dilution
of 10ul of above + 990ul of HT1
I can possibly load @ 8 pM which is decent.
By this way I wouldn't be exceeding 1mN NaOH in the final solution which is strictly recommended by Illumina but I'm still doubt full. Please let me know if I am right if any body has done/know similar approach.
Motivation: similar modifications in following paper and also some seqanswers posts
on page 18.2.22 says that in denaturation step, care should be taken not to exceed 1mM in final solution.