Blunting is the elimination of incompatible 3′ or 5′ overhangs for the promotion of blunt-end ligation. Several approaches may be used for DNA end blunting. Terminal unpaired nucleotides may be removed from DNA ends by using an enzyme with exonuclease activity, which hydrolyzes a terminal phosphodiester bond, thereby removing the overhang one base at a time. DNA fragments with 5′ overhangs may be blunted by filling in a recessed 3′ terminus with DNA polymerase in the presence of dNTPs. End removal or fill-in can be accomplished using a number of enzymes, including DNA Polymerase I Large (Klenow) Fragment (NEB #M0210), T4 DNA Polymerase (NEB #M0203) or Mung Bean Nuclease (NEB #M0250). Once blunted, DNA is universally compatible with other blunt-ended fragments and vectors.
Protocols for DNA Blunting
- Double Digest Protocol with Standard Restriction Enzymes
- Isolation of MBP-fusion protein using Amylose Magnetic Beads
- Loop-mediated Isothermal Amplification (LAMP)
- M13mp18 Phage Transfection
- Optimizing Restriction Endonuclease Reactions
- PCR Protocol for Phusion® High-Fidelity DNA Polymerase (M0530)
- Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using DNA Polymerase I, Large (Klenow) Fragment (M0210)
- Protocol for blunting ends by 3’ overhang removal and 3’ recessed (5’ overhang) end fill-in using T4 DNA Polymerase (M0203)
- Protocol for Digestion Prior to droplet digital PCR (ddPCR)
- Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
- Protocol for Phusion® High-Fidelity PCR Master Mix with GC Buffer
- Protocol for the Quick Blunting Kit (E1201)
- Protocol Phusion® High-Fidelity PCR Master Mix with HF Buffer
- Removal of Single-Stranded Extension Protocol using Mung Bean Nuclease (M0250)
Common Applications of Exonucleases and Endonucleases
Properties of Exonucleases and Endonucleases
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The first step in determining how your ends will be blunted is to determine if they are 5´ or 3´ overhangs. This tutorial will teach you how to identify what type of overhang you have, as well as which enzyme will blunt that end, and how.