New England Biolabs, M0345S, Exonuclease V (RecBCD)

Related Categories Exonucleases and Non-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme required to produce 1 nmol of acid-soluble deoxyribonucleotide from double-stranded DNA in 30 minutes at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X NEBuffer™ 4 Supplement with 1 mM ATP Incubate at 37°C 1X NEBuffer™ 4 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 1 mM DTT (pH 7.9 @ 25°C) Storage Buffer 50 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% Triton® X-100 pH 7.5 @ 25°C Heat Inactivation 70°C for 30 minutes Unit Assay Conditions 1X NEBuffer 4, 1 mM ATP with 0.15 mM sonicated duplex [ 3 H]-DNA. FAQ Q: What is the best reaction volume for removal of linear dsDNA? A: We recommend incubating a 30 µl reaction containing 10 units of Exonuclease V for 30 minutes at 37°C in order to remove 1 µg of linear dsDNA ranging from 100-1500 bp. One can estimate the amount of linear DNA present by agarose gel electrophoresis or nanodrop measurement. Treated DNA can be further purified by ethanol precipitation or spin-columns (e.g., Monarch PCR & DNA Cleanup Kit (NEB T1030). Recommended reaction setup (For removal of less than 1 µg/ul DNA): dsDNA x μl 10 x NEBuffer 4 3 µl 10 mM ATP 3 µl H2O 23-x µl Exonuclease V 1 µl Total 30 µl Q: Does isolated genomic DNA purified by different methods change the cleavage rate of Exonuclease V? A: Yes. Exonuclease V requires free-ends of ss/ds DNA to act on. Genomic DNA isolated from a spin column more easily digested, as compared to glass-rod spooled DNA. In general, 5 units of Exonuclease V is enough to digest 1 μg of gDNA (HeLa cell) which as been purified by a spin column, and 20 units of Exonuclease V can be used to digest 1 μg of glass-rod spooled E. coli gDNA. Q: Should I add additional ATP with Exonuclease V if I am scaling up my reaction? A: Yes. Exonuclease V requires ATP to cleave ss/ds DNA. However, greater than 1 mM ATP can cause inhibition. If more than 1 μg of DNA needs to be removed, one should increase all components proportionally, and incubate the reaction at 37°C for 30 min. Q: Does Exonuclease V (RecBCD) work in other NEBuffers? A: Yes. Exonuclease V has similar activity in NEBuffers 2, 3 and 4 (as long as 1 mM ATP is added). It is 50% active in NEBuffer 1. It also ~75% active in T4 DNA Ligase Reaction Buffer with no supplement of ATP. We recommend adding 1 mM ATP to the reaction if using Exonuclease V directly in a ligation reaction. Q: What is the best exonuclease to remove chromosomal DNA during a plasmid prep? A: Exonuclease V (RecBCD) removes residual chromosomal DNA after purification of a plasmid. Q: Which enzyme can distinguish between mitochondrial and genomic DNA and only remove gDNA? A: Exonuclease V (RecBCD) degrades linear ssDNA and dsDNA while preserving nicked and supercoiled plasmid DNA. 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. Q: Does NEB have an equivalent to Lucigen’s Plasmid-Safe? A: Exonuclease V is functionally equivalent to Plasmid-Safe and can be used to remove contaminating chromosomal DNA from plasmid preps to maintain double-stranded circular DNA.