Blunting protocol for NEB PCR Cloning Kit
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Reaction volume may be scaled up or down as necessary.
- 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 dH20: variable
Total volume: 25 µl
- 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.
* PCR generated DNA must be purified before blunting by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.
Restriction enzyme digested DNA can be blunted directly without purification. The Blunt Enzyme Mix has been optimized in Blunting Buffer, but is also active in NEBuffers 1.1, 2.1, 3.1, and CutSmart® Buffer in addition to NEBuffers 1-4, BamHI, EcoRI and DpnII unique buffers when supplemented with dNTPs and dithiothreitol. There is a small reduction in ligation fidelity in these buffers. Transformation efficiency is lowest in NEBuffer 1 and 1.1 where the total yield is about 50% of optimum.
- Immediately inactivate enzyme in the blunting reaction by heating at 70°C for 10 minutes.
- 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 room temperature.