Common Applications for Exonucleases and Endonucleases

Application Recommended Enzyme(s)
Removal of 3′ overhangs Quick Blunting Kit
T4 DNA Polymerase* + dNTPs
5′ overhang Fill in Treatment Quick Blunting Kit
T4 DNA Polymerase* + dNTPs
Klenow + dNTPs
Removal of oligonucleotides post PCR Exonuclease I
Removal of Chromosomal DNA in plasmid preparations Lambda Exonuclease
(Exonuclease I can be added to remove ssDNA generated by Lambda Exonuclease)

T5 Exonuclease
(Degrades linear ss + dsDNA, nicked DNA)

Exonuclease V (RecBCD)
(Degrades linear ss + dsDNA)
Removal of DNA in RNA preparations
DNase I
Chromatin Immunoprecipitation (ChIP) analysis
Micrococcal Nuclease
Generating ssDNA from linear dsDNA
If 5′ → 3′ polarity required (require one 5’-end with phosphate)
If 3′ → 5′ polarity required (require one end with 5’ overhang and one end with 3’overhang)
Best general choice

Lambda Exonuclease

Exonuclease III

Lambda Exonuclease



* T4 DNA Polymerase has a strong exo- activity.