Removal of Residual gDNA after Purification of Low Copy Plasmid using Exonuclease V (RecBCD)

  1. Transform an endA- strain (e.g. NEB 10-beta, NEB #C3019) with the BAC plasmid DNA and plate outgrowth onto a media plate with appropriate antibiotic. Incubate overnight at 30°C. BACs with CamR require reduced stringency selection. Chloramphenicol levels should be maintained between 10-15 μg/ml on the selective plate. Note: strains with an F’ plasmid are not compatible with BACs or miniF plasmids.
  2. Pick a colony, inoculate 10 ml LB + antibiotic, and incubate overnight at 30°C (200-250 RPM).
  3. Check OD600 nm (usually it will be around 4 O.D./ml of cells).
  4. Harvest 3 ml of the overnight culture and purify the plasmid DNA using the Monarch Plasmid Miniprep kit (NEB #T1010) following the recommended protocol.
  5. In the final elution step, elute the DNA with 30 μl of Monarch DNA Elution Buffer (NEB #T1016) (pre-heated to 50°C).
  6. To the eluted DNA, add 4 μl of NEBuffer 4 (10X), 4 μl of 10 mM ATP, and 2 μl of Exonuclease V (RecBCD). Mix reaction and incubate at 37°C for 1 hr.
  7. Heat-inactivate the Exonuclease V by incubating at 70°C for 30 min. The plasmid DNA is now ready for restriction enzyme digestion, PCR or transformation.
    Note: Typically 30-60 ng of single-copy plasmid can be purified from 3 ml of an overnight E.coli culture with (O.D. 600 nm = 4 O.D/ml)

    For more information, view our application note: Using Exonuclease V (RecBCD) to eliminate residual genomic DNA when purifying low copy plasmids with the Monarch® Plasmid Miniprep Kit