FAQ: How can I achieve optimal ligation of a 5’ phosphorylated ssDNA oligo to the 3’ end of an ssRNA substrate with a ssDNA or ssRNA splint using T4 RNA Ligase 2?

The reaction buffer included with this enzyme is optimized for nick ligation in dsRNA. To optimize the ligation of nicks in DNA/RNA hybrids, we recommend increasing the magnesium concentration from 2mM to 10mM in the reaction. Addition of 10-15% PEG8000 to the final reaction will further boost ligation efficiency.

The current buffer is good for nick ligation in dsRNA, but not for nick ligation in DNA/RNA hybrid.