Protocol for the Quick Blunting Kit (E1201)

1. Mix the following components in a sterile microfuge tube:

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.