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Old 02-22-2012, 09:59 AM   #1
pmonnahan
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Location: University of Kansas

Join Date: Jan 2012
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Default Excess Tween 20 in library for HiSeq

I recently prepared a library using the Multiplexed Shotgun Sequencing protocol (citation below) and sent the library off for sequencing on Illumina HiSeq. The library that I submitted was supposed to be at 10 nM in Qiagen EB buffer supplemented with 0.1% Tween 20. I received word that the library had failed to cluster on the flowcell.

After reviewing my notes, I noticed a large mistake...I added about ~100x too much Tween 20. The final solution was 10% instead of 0.1%. I have a feeling that this is probably what prevented clustering on the flowcell, but I can't find any conclusive info. Does anyone know if excess Tween 20 would interfere with HiSeq?

Thanks



Andolfatto, P., D. Davison, D. Erezyilmaz, T. T. Hu, J. Mast, T. Sunayama-Morita, and D. L. Stern. Multiplexed shotgun genotyping for rapid and efficient genetic mapping. Genome Research 21:610-617.
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Old 02-28-2012, 08:52 AM   #2
Rocketknight
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Default

Unfortunately, I'd say that very high Tween 20 would probably interfere with DNA binding to the flow cell. I don't think anyone would have done any definitive studies on this.

On the bright side, wasting thousands of dollars/euros/lives via minor errors is part and parcel of next-gen sequencing. Happens to us all at some point or another.
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