Plasmid safe atp dependent dnase kit

ZYCY10P3S2T Escherichia coli (System Biosciences, Palo Alto, CA, USA) were transformed with the original PPs of all four constructs (HDR-MC or HITI-MC, Cas9-AAVS1-MC, and Cas9-scrambled-MC), and viable colonies were selected using kanamycin plates. Colonies were picked 24 hours after transformation and grown in 6 ml of lysogeny broth (LB) with kanamycin for 6 hours at 37°C, followed by growth in terrific broth (TB) for 12 hours at 37°C. To induce expression of the phiC31 integrase for MC production via attB and attP recombination, 100 ml of LB broth together with 100 μl of 20% arabinose induction solution (System Biosciences, Palo Alto, CA, USA) and 4 ml of 1 N NaOH was added to the culture and grown for 5.5 hours at 30°C. An endotoxin-free maxi kit (Qiagen, Valencia, CA, USA) was used to purify both PP and MC. Following purification of the MC products, PP contamination was removed using the Plasmid Safe ATP-dependent DNase Kit (Epicentre, WI, USA), and the products were cleaned and concentrated using the Clean & Concentrator-25 Kit (Zymo Research, CA, USA).