Editing Genomic DNA from Bateria

Preparation of Genomic DNA from Bacteria

BASIC PROTOCOL: MINIPREP OF BACTERIAL GENOMIC DNA

Bacteria from a saturated liquid culture are lysed and proteins removed by digestion with proteinase K. Cell wall debris, polysaccharides, and remaining proteins are removed by selective precipitation with CTAB, and high-molecular-weight DNA is recovered from the resulting supernatant by isopropanol precipitation.

Materials

TE buffer (APPENDIX 2)<br/>10% sodium dodecyl sulfate (SDS)<br/>20 mg/ml proteinase K (stored in small single-use aliquots at -20C)<br/>5 M NaCl<br/>CTAB/NaCl solution<br/>24:1 chloroform/isoamyl alcohol<br/>25:24:1 phenol/chloroform/isoamyl alcohol (UNIT 2.1)<br/>Isopropanol<br/>70% ethanol

1. Inoculate a 5-ml liquid culture with the bacterial strain of interest. Grow in conditions appropriate for that strain (i.e., appropriate medium, drug selection, temperature) until the culture is saturated. This may take several hours to several days, depending on the growth rate.

2. Spin 1.5 ml of the culture in a microcentrifuge for 2 min, or until a compact pellet forms. Discard the supernatant.

3. Resuspend pellet in 567 ul TE buffer by repeated pipetting. Add 30 ul of 10% SDS and 3 ul of 20 mg/ml proteinase K to give a final concentration of 100 ug/ml proteinase K in 0.5% SDS. Mix thoroughly and incubate 1 hr at 37C.

The solution should become viscous as the detergent lyses the bacterial cell walls. There should be no need to predigest the bacterial cell wall with lysozyme.

4. Add 100 ul of 5 M NaCl and mix thoroughly.

This step is very important since a CTAB-nucleic acid precipitate will form if salt concentration drops below about 0.5 M at room temperature (Murray and Thompson, 1980). The aim here is to remove cell wall debris, denatured protein, and polysaccharides complexed to CTAB, while retaining the nucleic acids in solution.

5. Add 80 ul of CTAB/NaCl solution. Mix thoroughly and incubate 10 min at 65C.

6. Add an approximately equal volume (0.7 to 0.8 ml) of chloroform/isoamyl alcohol, mix thoroughly, and spin 4 to 5 min in a microcentrifuge.

This extraction removes CTAB-protein/polysaccharide complexes. A white interface should be visible after centrifugation.

7. Remove aqueous, viscous supernatant to a fresh microcentrifuge tube, leaving the interface behind. Add an equal volume of phenol/chloroform/isoamyl alcohol, extract thoroughly, and spin in a microcentrifuge for 5 min.

With some bacterial strains the interface formed after chloroform extraction is not compact enough to allow easy removal of the supernatant. In such cases, most of the interface can be fished out with a sterile toothpick before removal of any supernatant. Remaining CTAB precipitate is then removed in the phenol/chloroform extraction.

8. Transfer the supernatant to a fresh tube. Add 0.6 vol isopropanol to precipitate the nucleic acids (there is no need to add salt since the NaCl concentration is already high). Shake the tube back and forth until a stringy white DNA precipitate becomes clearly visible. At this point it is possible to transfer the pellet to a fresh tube containing 70% ethanol by hooking it onto the end of a micropipet that has been heat-sealed and bent in a Bunsen flame. Alternatively, the precipitate can be pelleted by spinning briefly at room temperature.

If no stringy DNA precipitate forms in the above step, this implies that the DNA has sheared into relatively low-molecular-weight pieces. If this is acceptable, i.e., if DNA is to be digested to completion with restriction endonucleases for Southern blot analysis, chromosomal DNA can often still be recovered simply by pelleting the precipitate in a microcentrifuge.

9. Wash the DNA with 70% ethanol to remove residual CTAB and respin 5 min at room temperature to repellet it. Carefully remove the supernatant and briefly dry the pellet in a lyophilizer.

10. Redissolve the pellet in 100 ul TE buffer.

This may take some time (up to 1 hr) since the DNA is of high molecular weight. 15 ul of this DNA will typically digest to completion with 10 U EcoRI in 1 hr, which is sufficient to be clearly visible on an agarose gel, or to give a good signal during Southern hybridization.



== 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]]