Editing Genomic DNA from Bateria (section)

== Protocol 2 ==

<br/>Preparation of Bacterial Genomic DNA<br/>1) Inoculate a 5 ml culture and incubate overnight<br/>2) Transfer in hemolysis tube and spin the cells down until a compact pellet forms. Discard&nbsp; the supernatant. &nbsp;<br/>3) Resuspend the pellet by&nbsp; repeating pipetting in a&nbsp; mix of 567μl TE buffer and&nbsp; 5μl RNAse<br/>A. Add 15 μl 10% SDS and 4 μl proteinase K (18 mg.mL-1). Mix thoroughly and incubate 1h at 37°C or a few minutes at 65°C until all the cells are lysed.<br/>The solution should become viscous as the detergent lyses the bacterial cell walls.<br/>4) Add 100μl of 5M NaCl and mix thoroughly.<br/>This&nbsp; step&nbsp; is&nbsp; very&nbsp; important&nbsp; since&nbsp; a&nbsp; CTAB-nucleic&nbsp; acid&nbsp; precipitate&nbsp; will&nbsp; form&nbsp; if&nbsp; salt concentration drops below 0.5M at room temperature ( Murray and Thompson, 1980). The aim here is to remove cell wall debris, denaturated protein and polysa ccharides complexed<br/>with CTAB, while the nucleic acids in solution.<br/>5) Add 80μl of CTAB/NaCl (10% w/v; 0.7M) solution. Mix thoroughly and incubate&nbsp; 10min at 65°C.<br/>6) Add&nbsp;&nbsp; approximately&nbsp;&nbsp; equal&nbsp;&nbsp; volume&nbsp;&nbsp; of&nbsp;&nbsp; chloroform/isoamyl&nbsp;&nbsp; alcohol&nbsp;&nbsp; (0.7-0.8ml ),&nbsp;&nbsp; mix thoroughly and spin 4-5 min in a centrifuge. &nbsp;<br/>The&nbsp;&nbsp; extraction&nbsp;&nbsp; removes&nbsp;&nbsp; CTAB-protein/polysaccharides&nbsp;&nbsp; complexes.&nbsp;&nbsp; A&nbsp;&nbsp; white&nbsp;&nbsp; interface should be visible after centrifugation.<br/>7) Remove aqueous, viscous supernatant to a fresh Falcon tube, leaving&nbsp; the interface behind.<br/>Add&nbsp; an&nbsp; equal&nbsp; volume&nbsp; of&nbsp; phenol/chloroform/isoamyl&nbsp; alcohol,&nbsp; extract&nbsp; thoroughly&nbsp;&nbsp; and&nbsp; spin for 5 min.<br/>With&nbsp; some&nbsp; bacterial&nbsp; strains&nbsp; the&nbsp; interface&nbsp; formed&nbsp; after&nbsp; chloroform&nbsp; extraction&nbsp; is&nbsp; not compact&nbsp; enough&nbsp; to&nbsp; allow&nbsp; easy&nbsp; removal&nbsp; of&nbsp; the&nbsp; supernatant.&nbsp; In&nbsp; such&nbsp; cases,&nbsp; most&nbsp; of&nbsp; the interface&nbsp; cans&nbsp; be&nbsp; fished&nbsp; with&nbsp; a&nbsp; sterile&nbsp; toothpick&nbsp; before&nbsp; removal&nbsp; of&nbsp; the&nbsp; supernatant.<br/>Remaining CTA precipitates is then removed in a phenol/chloroform extraction.<br/>8) Add 1 volume&nbsp; of&nbsp; isopropanol, shake and&nbsp; spin. Wash&nbsp; with 70% ethanol, dry and dissolve the pellet in 50μl Tris pH = 7 or water. 
[[Category:Protocols]]