How is the concentration and the quality of the DNA samples before starting the protocol Nextera XT (Illumina)?

Concentration was 250ng/ul with 260/280 and 260/230 around 1.9 and 1.7.

The stock with 250ng/ul was diluted to 0.2 ng/ul in Elution Buffer(tris) that is it was diluted manyfold based based on Qubit readings at each dilution. So if there was any contamination, the contaminants were also diluted multiple times.

Still, we tried cleaning up our samples with Ampure XP beads, we obtained same results.

But you run the original sample on the gel?

I have attached the gel picture. It looks like a good quality DNA on gel.

There is a smire inside the sample. This is not so much good for the quality.

Nikhil Bhalla your DNA sample present a smire up the band. This means that your sample is not good. Is possible that your DNA extraction is not perfect.

How much DNA you load inside the gel? And how was the DNA concentration?

Hi georgia,

We have tried it with another gDNA sample which has worked in past perfectly, however it did not work this time.

We have also tried it with a Qia Purified Plasmid DNA but with no success.

Why do you use the kit for purified plasmid DNA?

Do you try to amplify of 16S thorugh a normal PCR?

Giorgia Pertile Yes, but not 16S, we have used another gene which is species specific in a conventional PCR using gDNA with Phusion polymerase. It amplified perfectly.

Try to test the 16S on your DNA sample in normal PCR to check the DNA.

Do you are sure about the specificty of this specific primer?

Try to check the specificity of the primer through the BLAST. Take the sequence of your foward primer and BLAST inside NCBI vs all the database.

Thanks Giorgia.

Contacted Illumina's tech support, will inform you about the problem. Thanks for your patience and valuable suggestions.