Bacterial DNA Isolation and Purification

Bacterial cell pellets equal to 4 OD680 were resuspended in 400 μl of 50 mmol/L Tris/50 mmol/L EDTA，pH 8.0，by gentle vortex.The cells were lysed by the addition of 8 μl of 50 mg/ml lysozyme solution in TE，with subsequent incubation at 37℃for 30 min.Following cell lysis，4μl of 10%SDS and 8μl of 14—15 mg/ml proteinase K solution were added to each tube，and the cellular proteins were digested at 50℃for 30 min.Proteinase K was then heat-inactivated at 75℃for 10 min.Next，RNA was removed by the addition of 2 μl of 10 mg/ml RNase solution，followed by incubation at 37℃for 30 min.DNA was subsequently extracted by adding 425 μl of phenol：chloroform：isoamyl alcohol（25∶24∶1），vortexing the tube vigorously and intermittently for 20 s，and sedimenting the tube for 5 min at 10，000 rpm（10，000 g），4℃.The upper aqueous phase was carefully transferred to a fresh micro centrifuge tube using a wide-opening pipet tip（a regular tip cut about 0.5 cm from the end with a clean razor blade）.DNA was further extracted by adding 400 μl chloroform，vortexing the tube vigorously and intermittently for 20 s，and sedimenting the tube for 5 min at 10，000 rpm（10，000 g），4℃.The upper aqueous phase was carefully transferred to a fresh microcentrifuge tube using a wide-opening pipet tip.The contents of the tube were subjected to final sedimentation for 5 min at 10，000 rpm（10，000 g），4℃，and the resulting clear supernatant，containing purified total bacterial DNA（genomic DNA，or gDNA，and plasmid DNA，or pDNA），was transferred to a clean tube.Note that thorough vortexing and use of wide-opening pipet tips is critical during the phenol/chloroform extraction steps to obtain a condensed interface，and to avoid contaminating the upper viscous aqueous phase with denatured proteins and polysaccharide from the interface.If the upper aqueous phase is still too viscous for pipetting，repeat the vortexing and spinning steps in the phenol/chloroform extraction.(https://www.daowen.com)