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Old 04-20-2018, 04:38 PM   #1
joyeakley
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Location: San Diego

Join Date: Apr 2018
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Default using P5 as a sequencing primer

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!
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