Department of Biological Sciences,

1000 Hilltop Circle

Baltimore, MD 21250

Preparation of Genomic DNA from Bacteria- using Phase Lock Gel^TM

^{(Modified from Experimental Techniques in Bacterial Genetics, Jones and Bartlet, 1990)}

Materials: see Solutions for Recipes

	TE buffer
	10% (w/v) sodium dodecyl sulfate (SDS)
	20 mg/ml proteinase K
	phenol\chloroform (50:50)
	isopropanol
	70% ethanol
	3M sodium acetate pH 5.2
	Phase Lock GelTM (Eppendorf-Brinkmann) (http://www.eppendorf.com/phaselockgel/en/phase_1.html)
	Grow E. coli culture overnight in rich broth. Transfer 2 ml to a 2-ml micro centrifuge tube and spin 2 min. Decant the supernatant. Drain well onto a Kimwipe. Resuspend the pellet in 467 μl TE buffer by repeated pipetting. Add 30 μl of 10% SDS and 3 μl of 20 mg/ml proteinase K, mix , and incubate 1 hr at 37 ° C. Add an equal volume of phenol/chloroform and mix well but very gently to avoid shearing the DNA by inverting the tube until the phases are completely mixed. CAUTION: PHENOL CAUSES SEVERE BURNS, WEAR GLOVES GOGGLES, AND LAB COAT AND KEEP TUBES CAPPED TIGHTLY. Carefully transfer the DNA/phenol mixture into a Phase Lock GelTM tube (green) and spin at 12,000 RPM for 10 min. Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform. Again mix well and transfer to a new Phase Lock GelTM tube and spin 10 min. Transfer the upper aqueous phase to a new tube. Add 1/10 volume of sodium acetate. Mix. Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates. Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed end). Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sec. Resuspend DNA in at least 200 μl TE buffer. Complete resuspension may take several days. Store DNA at 4 ° C short term, -20 or -80 ° C long term. After DNA has dissolved, determing the concentration by measuring the absorbance at 260 nm.