Application Features

Prepare sheared, nebulized or restriction enzyme digested DNA for blunt-ended ligation into a plasmid, cosmid, fosmid or BAC vector

Prepare PCR products for efficient blunt-end cloning

Companion Products

• Quick Blunting™ and Quick Ligation™ Kits
• Quick T4 DNA Ligase
• Monarch® Plasmid Miniprep Kit 
• Monarch® DNA Gel Extraction Kit
• Monarch® PCR & DNA Cleanup Kit (5 μg)

1. PCR generated DNA must be purified before blunting by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.
2. 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 1,2,3 and 4, as well as 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 where the total yield is about 50% of optimum.
3. ATP is not necessary for T4 Polynucleotide Kinase activity in the kit. The dATP and dTTP in the dNTP mix act as phosphate donors.
4. The blunted reaction must be purified prior to phosphatase treatment by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.

1. Which ligase is recommended to ligate DNA treated with the Quick Blunting Kit?
2. Can I use regular ligase to ligate my blunted product?
3. I've blunted my DNA and now need to dephosphorylate the reaction with Antarctic Phosphatase. Do I need to purify the reaction?
4. Does the PCR product need to be purified before blunting with the Quick Blunting Kit? What methods can be used to purify the product?
5. I've digested my DNA and now want to blunt directly without purifying, can I add the blunting reagents directly to the restriction digest?
6. I've blunted my sonicated gDNA for 15 minutes instead of the recommended 30 minute incubation time, will the reaction still work?
7. I've accidentally skipped the heat-kill step after the blunting reaction. Will the ligation still work?

1. Blunting protocol for NEB PCR Cloning Kit
2. Protocol for the Quick Blunting Kit (E1201)
3. Quick Ligation Protocol (M2200)
4. Transformation Protocol

• Blunting Selection Chart

• EpiMark® Methylated DNA Enrichment Kit Troubleshooting Guide

• NEBcloner

• E1201S_L_v1

• E1201S_L_v1_0101801
• E1201L_v1_10013484
• E1201L_v1_10013485

• 10X Blunting Buffer
• Blunting Enzyme Mix
• Deoxynucleotide Solution Mix (1 mM)