NEB Library fragment size erro

[aurbano]

Hello everyone!

I have a question, I´m using NEB ultra prep DNA for my amplicom libraries (total size 150+120).
In the last step, when finish the PCR for the enrichment of adaptor ligated DNA I chek it in agarosa gel and I see a smear with a band in 600-1000 pb. This band is diferent in each library, some library 700, anothe 800...

I run a qPCR for quantification the librarys and I obtained very mixed result, from 1,5 nM to 1600nM...

The last time I sequenced the same library i obteined underclustering. I run in miniseq and it was 33k/mm2 and I think that it is the problem.


somebody could help me??

Thank you

[nucacidhunter]

Library size issue need to be sorted out first. Larger than expected size points to possible PCR overcycling. Also, to convert qPCR data to nM you need to determine size of library accurately which is not possible by gel.