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Old 10-03-2013, 01:21 AM   #5
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Location: Basel (Switzerland)

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Originally Posted by Sully View Post
I am having similar troubles at the tagmentation step and I hope someone can make comments on this; I have checked the quality of my gDNA samples on gel, obtained 260/280 and 260/230 ratios on nanodrop and quantified them using Qubit, cross checked the concentration with QuanIT standard curve, also. But I consistently get bioanalyzer profile with the peak centered around 1.2 kb under standard conditions (5 min, 55 C). Interestingly, protracted incubation up to ten min produced very similar profiles, too. I then started with 25 ng total gDNA, which improved the profile but the peak is sitting around 1 kb. I have also included control DNA (lambda DNA which comes with another commercial kit and quantified by the manufacturer) and obtained the same exact profile. My thermocyler and pipettes are calibrated annually. I am almost ready to conclude the nextera enzyme I have received is from a bad lot. What else I may try before contacting Illumina? Thank you.
it could be that the enzyme comes froma bad lot but why using so much for tagmentation? 25 ng is not necessary! Reducing the input will help to give shorter libraries and the data quality won´t be affected. Prolonged incubation at 55 degrees, like it was reported in the previous post, doesn´t work because when the transposase ligates adaptors it becomes inactive. So, if after 5 min there is no more transposes loaded with oligos, even incubating 1 hour won´t change the size distribution of the library.
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