sequencing low concentrated amplicons on MiSeq

[sriman]

Hello,

I came across acockburn's suggestion on using the protocol published in the Supplementary Protocol 12: Modified hybridization buffers of the following paper: A large genome center's improvements to the Illumina sequencing system for sequencing low concentrated libraries.

My question is on preparing the neutralization buffer, it is not clear to me whether I should add the 1 M Tris-HCl pH7.3 in the neutralization buffer or is this only added to the HT1?

Please let me know if anyone has prepared this buffer before.

Thanks a lot!!

[JakobHedegaard]

Hi,
If we happen to have libraries of low concentration, we denature with 0.2 M NaOH, add an equal volume of 200 mM Tris-HCl, pH 7, and adds HT1 to reach 20 pM.
An example: a library is 230 pM. Take 10 µL library and add 10 µL 0.2 M NaOH to denature. Then add 10 µL 200 mM Tris-HCl, pH 7, to neutralize. Finally, add 85 µL HT1 to make a 20 pM solution.
/Jakob