SEQanswers - View Single Post - Next-seq starting library concentration for library denaturation

huguesparri · 10-17-2016, 07:38 AM

Hi Irene,
First of all, I don't have experiment with the nextseq system. We have a Hiseq2500 but I suppose the issue and solutions are the same.
What we usually do when our library concentration is too low is to add a neutralization step after the NaOH denaturation: we add 0.1N HCl to neutralize the NaOH.
The point is to be sure that we don't end up with a NaOH concentration higher than 0.001 N(1mM) in your final library dilution (the one you're going to cluterise with). Failing to do so could inhibit the library hybridizaiton on your flow cell and give you a lower cluster density.

10-17-2016, 07:38 AM	#2
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	Hi Irene, First of all, I don't have experiment with the nextseq system. We have a Hiseq2500 but I suppose the issue and solutions are the same. What we usually do when our library concentration is too low is to add a neutralization step after the NaOH denaturation: we add 0.1N HCl to neutralize the NaOH. The point is to be sure that we don't end up with a NaOH concentration higher than 0.001 N(1mM) in your final library dilution (the one you're going to cluterise with). Failing to do so could inhibit the library hybridizaiton on your flow cell and give you a lower cluster density.