Hello All -
I'd like to use P5 (engrafted or exogenous) as a sequencing primer for a library consisting of ~200 synthetic sequences 183bp long, including the full length P5 and P7 ends. Sanger sequencing shows the structure is correct, and the pool is the correct size on a gel. There were lots of clusters on a miniSeq run, but when the P5 29-mer (Tm 62C) was used as a custom Read 1 primer (from the correct cartridge position), there were no reads and the run failed after 25 cycles, consistent with a registration failure. The instrument runs other libraries fine, but other custom Read 1 primers have also failed in previous runs.
Does anyone have success with custom Read 1 on miniSeq? Can I use P5 as a custom sequencing primer? If so, should it be added exogenously or should it be the engrafted primer?
Many thanks for any suggestions!
I'd like to use P5 (engrafted or exogenous) as a sequencing primer for a library consisting of ~200 synthetic sequences 183bp long, including the full length P5 and P7 ends. Sanger sequencing shows the structure is correct, and the pool is the correct size on a gel. There were lots of clusters on a miniSeq run, but when the P5 29-mer (Tm 62C) was used as a custom Read 1 primer (from the correct cartridge position), there were no reads and the run failed after 25 cycles, consistent with a registration failure. The instrument runs other libraries fine, but other custom Read 1 primers have also failed in previous runs.
Does anyone have success with custom Read 1 on miniSeq? Can I use P5 as a custom sequencing primer? If so, should it be added exogenously or should it be the engrafted primer?
Many thanks for any suggestions!