New England Biolabs, M0293L, Exonuclease I (E. coli)

Note: See also Exo-CIP™ Rapid PCR Cleanup Kit ( Related Categories Exonucleases and Non-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme that will catalyze the release of 10 nmol of acid-soluble nucleotide in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X Exonuclease I Reaction Buffer with 0.17 mg/ml single-stranded [ 3 H]-DNA. Reaction Conditions 1X Exonuclease I Reaction Buffer Incubate at 37°C 1X Exonuclease I Reaction Buffer 67 mM Glycine-KOH 6.7 mM MgCl2 10 mM β-ME (pH 9.5 @ 25°C) Usage Concentration 10,000 units/ml Storage Buffer 10 mM Tris-HCl 100 mM NaCl 5 mM β-ME 0.5 mM EDTA 100 µg/ml BSA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 80°C for 20 minutes FAQ Q: Can Exonuclease I be used to remove primers from amplification reactions? A: Exonuclease I degrades excess single stranded primer oligonucleotides from a reaction mixture containing double stranded products. Q: Will Exonuclease I degrade RNA? A: No. Q: Can Exonuclease I be used to blunt DNA? A: No. Short ends are not a substrate. Use DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) to fill in 5' overhangs (also called 3' recessed ends) and chew back 3' overhangs or use Mung Bean Nuclease (NEB# M0250) to chew back 3' or 5' overhangs. Note: 3' overhangs can not be filled in. Q: Can Exonuclease I be heat inactivated? A: Yes. Heat inactivate at 80°C for 20 minutes. Q: Will Exonuclease I work in other buffers? A: Yes. For common applications Exonuclease I will work in NEBuffers 1-4 and in the polymerase buffers. Q: Can Exonuclease I be used with a double stranded exonuclease to clean up plasmid preparations? A: Exonuclease I can be used with Lambda Exonuclease (NEB# M0262) to clean up plasmid preps. Exonuclease III (NEB# M0206) and T7 Exonuclease (NEB# M0263) will also work, but will damage nicked plasmids. Although Exonuclease I can be used, we recommend using Exonuclease V (RecBCD) (NEB #M0345) to remove chromosomal DNA after plasmid prep (see our Application Note: Using Exonuclease V (RecBCD) to Eliminate Residual Genomic DNA When Purifying Low Copy Plasmids with the Monarch® Plasmid Miniprep Kit.