What are the reasons why Nextera XT library prep kit fail?

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Michail Yekelchyk

Hi,

First of all, as mentioned above, check your DNA. Measure the concentration with Qubit (or analogues) and check the size distribution of Bioanalyzer / gel. Nextera XT is nicely working starting from roughly 0.1 ng of total DNA, and you can adjust the number of PCR cycles to get more library.

Usually Nextera XT is a very reliable kit, I made a lot of libraries with it. Though, once I had a problem due to expired "Amplicon Tagment Mix". There is a Tn5 transposase inside, which cuts the double-stranded DNA and simultaniousely inserts 2 adapters for PCR. So when it expires (or with wrong storage conditions), the efficiency is dropping. I even had a feeling that it started to insert only 1 adaptor instead of 2. Anyway, when we bought a new kit, it worked perfectly again.

Best,

Michail

Amor Ii Damatac

Giorgia Pertile Hi! Yes, we checked before lib preparation and since it's a PCR product we are pretty sure that there is enough DNA. Thank you!

Michail Yekelchyk Hello! The DNA was good. The kit was also perfectly working with the other samples. I will try to increase the number of PCR cycles. Thank you!