Protocol for the Quick Blunting Kit (E1201)
|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|
|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.