Hi all,
I'm having troubles with a MiSeq run. I used a Multiplicom NGS kit, specific for two genes. I followed the protocol and produced 4nM libraries. I pooled these libraries and than denaturated the pool (5ul pool + 5ul NaOH 0.2N fresh made) for 5 minute. I added HT1 buffer to 20 pM concetrantion, than diluted again to 12 pM and loaded this pooled diluted library togheter with 5% PhiX equimolar. I loaded the 3 custom primers requested by the protocol (read1, read2 and index) at the appropriate concentration. I started the run with correct sample sheet but it stopped before the first cycle, it seems for the impossibility to form clusters. The NGS kit I used was previously used in the lab without problems and I've tried to quantify libraries with tapestastion and the images I see are in agreement with the correct lenght of my libraries. I can't understand what went wrong and I'm wondering if can be a reagent issue. In few days I will reload the libraries, so a would like to understand why the instrument (MiSeq) stopped so early.
Could someone tell me his experience) Thank you in advance
I'm having troubles with a MiSeq run. I used a Multiplicom NGS kit, specific for two genes. I followed the protocol and produced 4nM libraries. I pooled these libraries and than denaturated the pool (5ul pool + 5ul NaOH 0.2N fresh made) for 5 minute. I added HT1 buffer to 20 pM concetrantion, than diluted again to 12 pM and loaded this pooled diluted library togheter with 5% PhiX equimolar. I loaded the 3 custom primers requested by the protocol (read1, read2 and index) at the appropriate concentration. I started the run with correct sample sheet but it stopped before the first cycle, it seems for the impossibility to form clusters. The NGS kit I used was previously used in the lab without problems and I've tried to quantify libraries with tapestastion and the images I see are in agreement with the correct lenght of my libraries. I can't understand what went wrong and I'm wondering if can be a reagent issue. In few days I will reload the libraries, so a would like to understand why the instrument (MiSeq) stopped so early.
Could someone tell me his experience) Thank you in advance
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