Can anyone help me with this Urea-PAGE gel issue?

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Fedor Kouzine

Without protocol it is difficult to tell for sure. However, I guess that some of you oligos could form intermolecular or intramolecular structures. Urea is fully effective as denaturant only at high temperature (optimum is around 50-55 C). That is why gel need to be pre-run.

Louis Cicchini

Probably a secondary structure. Make sure to boil your DNA in a denaturing buffer before loading

Lakshani Perera

Hi Fedor,

Thank you for the response. After adding formamide I do not boil my DNA and directly run on PAGE gel. But I do pre run the gel at 400 V for 10-15 min.