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Old 03-18-2016, 05:10 PM   #1
Junior Member
Location: Texas

Join Date: Aug 2015
Posts: 2
Unhappy Nextera XT tagmentation problem

Hello all,
I need some help with Nextera XT library prep from bacterial genome.
DNA were extracted using QIAmp kit with QIAcube HT robot and measured the DNA by absorbance and fluorescence.
I am following the Illumina protocol strictly and checking the library after PCR cleaning step to decide to move forward or start over the tagmentation & PCR.

I had few samples to start over this time, but I am now stuck with tagmentation step for few days.
I suspected inhibitor contamination or wrong DNA concentration, so cleaned the DNA with Zymo gDNA cleaning kit and eluted with water to avoid EDTA. DNA was measured by absorbance and fluorescence again to make sure the concentration. I confirmed the genomic DNA by running on QIAxcel.

Brand new TD buffer and ATM buffer was used. After tagmentation (55 C, 5min), no tagmentation was going on confirming by QIAxcel.

Does anyone had same experience? The DNA were all extracted under same condition at the same time, so why does this difference happens?

In previous thread, it was recommended to clean with Mo-bio kit, so I will try that too.

Any help or suggestions will be appreciated! I am now thinking to re-extract the DNA.
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