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Old 09-18-2013, 07:44 AM   #4
Sully
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Location: Maryland

Join Date: Feb 2013
Posts: 1
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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.
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