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Illumina library preparation - adaptor ligation
Hi all, I am quite new to Illumina amplicons library preparation. I am having issue regarding adaptors ligation.
I figure out the problem having double bands after my SPRI cleaning of PCR enrichment steps. I have a really low amount of PCR products compare to my first PCR and a high concentrations of adaptors adaptors systems. However, I am still able to get a proper qPCR with a Ct average value around 6 to 9.
I tried changing the final extension time of my first PCR whitout significant changes. I also tried renewing all my stocks. I tried with different concentrations of adaptors and I tried with different concentrations of PEG in my SPRI. Still, I have always double bands and really low PCR products after my PCR enrichment tests.
My samples will be pooled with other projects. These projects all worked fine. The main difference is that their targeted sequence is around 200 to 300 bp while mine is 500 bp.
To anyone this issue occured?
I figure out the problem having double bands after my SPRI cleaning of PCR enrichment steps. I have a really low amount of PCR products compare to my first PCR and a high concentrations of adaptors adaptors systems. However, I am still able to get a proper qPCR with a Ct average value around 6 to 9.
I tried changing the final extension time of my first PCR whitout significant changes. I also tried renewing all my stocks. I tried with different concentrations of adaptors and I tried with different concentrations of PEG in my SPRI. Still, I have always double bands and really low PCR products after my PCR enrichment tests.
My samples will be pooled with other projects. These projects all worked fine. The main difference is that their targeted sequence is around 200 to 300 bp while mine is 500 bp.
To anyone this issue occured?