Protocol needs refining

Isolation of E. coli Genomic DNA
communicated by Dr. Jon Visick

1. Grow a 5-ml culture in LB (or a richer broth like Terrific Broth if desired for higher cell concentration).
2. Pellet cells (5 min. at 5000 rpm in a typical Sorvall) and resuspend in 250 ul of 50 mM Tris pH8/50 mM EDTA and transfer to a microfuge tube.
3. Freeze the suspension at -20° C, then add 25 ul of 10 mg/ml ysozyme in 0.25 M Tris pH 8 (freshly made up). Let stand at room temperature with occasional mixing until just thawed, then place tube on ice for 45 min.
4. Add 50 ul of STEP solution (0.5% SDS, 50 mM Tris pH 7.5, 0.4 M EDTA, with proteinase K powder added to a final concentration of 1 mg/ml immediately before use), mix well and place in a 50° C water bath for 60 min, mixing (gently) occasionally.
5. Add 300 ul of saturated, Tris-buffered phenol (pH 8--be sure it's not acidic, or the DNA will be lost into the phenol phase!) and mix by inverting 30 times. Don't vortex, as this can shear the DNA.
6. Spin 5 min. in a microfuge to separate layers. Transfer the top, aqueous phase containing the DNA to a clean tube. If too much protein from the interface is transferred, the phenol step can be repeated.
7. Add 0.1 volume of 3 M sodium acetate and mix without vortexing.
8. Add 2 volumes of 100% ethanol and mix; you may be able to see a "glob" of DNA if there's enough of it (definitely you'll see it with larger culture volumes).
9. Spin 10 min. in microfuge to precipitate DNA. Remove ethanol thoroughly; you may want to dry under vacuum to facilitate redissolving the DNA.
10. Add 250 ul of 50 mM Tris pH 7.5, 1 mM EDTA, 200 ug/ml RNAse A (see note below). DNA must be dissolved completely; at this point I usually let it stand overnight at 4° C.
11. Some people use the DNA at this stage; if you're going to do so, it's probably best to redissolve it in only about 100 ul. (Though if you get a lot of DNA you may have to add more to get it dissolved.)

Alternatively, you can remove the RNAse by adding an equal volume of chloroform, mixing and centrifuging as with the phenol, reprecipitating with sodium acetate and ethanol and again spinning down the DNA. Then redissolve in about 100 ul of 50 mM Tris pH 7.5, 1 mM EDTA (I do another overnight redissolving step here).

Note on RNAse: If you don't have any made up, you can easily make DNAse-free RNAse by dissolving RNAse A in 25 mM Tris pH 7.5 at 5 mg/ml and boiling for 5 minutes to destroy DNAse. Then add an equal volume of 40% glycerol and 0.5 voumes of 0.5 M NaCl. The resulting 2 mg/ml stock is stable and can be stored at -20° C indefinitely.

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