- 0.003 mg/mL of 217-bp linear DNA with Ava II cohesive ends
- 0.006 mg/mL HU protein in 50 mM HEPES, pH 7.5
- 50 mM potassium glutamate
- 10 mM MgCl2
- 1 mM ATP in a 150 mL reaction vial
The reaction mixture was incubated for 30 minutes at 30 °C, after which time the end-joining reaction was initiated with the addition of 2000 U/ml T4 DNA ligase (New England Biolabs).
The reaction was terminated after 30 minutes by transferring the contents of the reaction vial to a Centricon-100 ultrafiltrator and successively washing with 300 mL solutions of 2 M and 0.1 M NaCl, respectively.
