Question: (Closed) Miseq loading with low concentration library
gravatar for rohan
6.1 years ago by
United States
rohan100 wrote:

Hi there,

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

Improved Protocols for Illumina Sequencing

on page 18.2.22 says that in denaturation step, care should be taken not to exceed 1mM in final solution.

ADD COMMENTlink written 6.1 years ago by rohan100

Hello rohan!

We believe that this post does not fit the main topic of this site.

BioStar is a site for bioinformatics questions, not questions related to sequencing in general. You should probably post this on the Illumina board at SeqAnswers:

For this reason we have closed your question. This allows us to keep the site focused on the topics that the community can help with.

If you disagree please tell us why in a reply below, we'll be happy to talk about it.


ADD REPLYlink written 6.1 years ago by User 5913k
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