MiSeq newbie / Library prep questions

[elenstea]

Hello everyone. I am using MiSeq from Illumina using NEXTERA DNA kit. I have already performed 2 runs (first run with 2X150 v.2 went super great , the second run with 2X75 v.3 which was not so good, very few clusters , but great quality). So I have some questions concerning library preparation:

1. Illumina recommends storing a library at +4 for a maximum of 7-10 days. As we are new users we have difficulties in library preparation, but we do have some excellent libraries (which we would like to pool, but this means storing, till the next successful library prep). Has anyone stored libraries at -20 or -80 with good results? and for how long were they stored?

2. Although Nextera recommends only QUBIT for quantification, we also use real time PCR, but with great discrepancies between the two methods. Of course real-time quantifies only for dsDNA with adapters which explains the discrepancies. But when we used the real time quantification (which is supposed to be more accurate) we had very low cluster generation. Did anyone else notice the same thing and would you try to "overcluster" (loading more than calculated) on purpose?

[Nikvailo]

Recently, we had difficulty related with qPCR-Qubit inconsistency. However, when we used qPCR results and prepare last library, we got over-clustered flow cell. In the second run, we used Qubit and Qubit gave higher concentrations. However, the pool that prepared according to Qubit gave excellent results. We are planning to use only Qubit for our next projects.

[elenstea]

thank you