Protocol for the Quick Blunting Kit (E1201)
| AMOUNT | |
| Purified DNA (up to 5 μg) | 1–19 μl |
| 10X Blunting Buffer | 2.5 μl |
| 1 mM dNTP Mix | 2.5 μl |
| Blunt Enzyme Mix | 1.0 μl |
| Sterile water | variable |
| Total volume | 25 μl |
2. Reactions containing restriction enzyme digested DNA are incubated at room temperature for 15 minutes. Reactions with sheared/nebulized DNA or PCR products are incubated at room temperature for 30 minutes.
3. Immediately inactivate enzyme in the blunting reaction by heating at 70°C for 10 minutes.
4. Proceed directly to the ligation step using the Quick Ligation Kit (NEB #M2200) or standard T4 DNA Ligase (NEB #M0202). Blunt ligation reactions using standard T4 DNA Ligase should be incubated overnight at 16°C.
