Are you sure of your quantification and quality of DNA?

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

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!