Hi all,
I’m having trouble generating an “optimal” library for sequencing.
I am using a human cell line derived from an adrenocortical tumor (from ATCC).
I have tried 12.5, 25, 50, and 100k cells with 2.5ul Tn5 (Illumina).
I've tried lysing with and without Digitonin, no difference.
qPCR determined that I needed ~5-7 extra cycles of amplification.
Qiagen cleanup was not removing the primer, and I had large peaks at ~2000 or bigger, so I switched to AMPure beads (double sided purification, first 0.5X then 1.8X)
I never seem to get those nice big peaks around ~<200, 400 just a blip. See pics below.
Does anyone have any ideas for improving the efficiency of my library amplification?
Thanks
Chris
I’m having trouble generating an “optimal” library for sequencing.
I am using a human cell line derived from an adrenocortical tumor (from ATCC).
I have tried 12.5, 25, 50, and 100k cells with 2.5ul Tn5 (Illumina).
I've tried lysing with and without Digitonin, no difference.
qPCR determined that I needed ~5-7 extra cycles of amplification.
Qiagen cleanup was not removing the primer, and I had large peaks at ~2000 or bigger, so I switched to AMPure beads (double sided purification, first 0.5X then 1.8X)
I never seem to get those nice big peaks around ~<200, 400 just a blip. See pics below.
Does anyone have any ideas for improving the efficiency of my library amplification?
Thanks
Chris
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