Hi,
I am trying to multiplex on the GAII with homemade PE adapters (incl 5nt barcodes) and all I see after cloning my libraries are adapter multimers. Each fragment contains the sequences for hybridization to the flowcell, so PCR is working (Im using Phusion), but my insert DNA is nowhere to be found. I am following the Illumina Protocol with some modifications suggested by Craig et al and Quail et al (sonication, double size selection, SPRI PCR cleanup etc). I designed my adapters to be identical to the Illumina PE adapters with the barcode tacked on the 3' end and a T overhang. I havent modified them in any special way, just HPLC purified. Any suggestions?
I am trying to multiplex on the GAII with homemade PE adapters (incl 5nt barcodes) and all I see after cloning my libraries are adapter multimers. Each fragment contains the sequences for hybridization to the flowcell, so PCR is working (Im using Phusion), but my insert DNA is nowhere to be found. I am following the Illumina Protocol with some modifications suggested by Craig et al and Quail et al (sonication, double size selection, SPRI PCR cleanup etc). I designed my adapters to be identical to the Illumina PE adapters with the barcode tacked on the 3' end and a T overhang. I havent modified them in any special way, just HPLC purified. Any suggestions?
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