ORS GENOMIC DNA ISOLATION

grow 5 mls late-log in rich medium
cent. 5 kg x 10'
resuspend in 4.5 ml TS
add .5 ml fresh 1% sarkosyl, vortex 15''
cent 10 kg x 10'
resuspend in .4 ml TES
add .35 ml sucrose mix
ice x 20'
add .15 ml lysozyme (5 mg/ml, made fresh)
add 30 ul of .5 M EDTA
ice x 20'
add .6 ml 10% sarkosyl
mix slowly
incubate 60 C x 30' with mixing until lysis is evident
extract with 1 ml phenol (10 kg x 20')
extract with 1 ml CHCl3 (10 kg x 20')
adjust aqueous phase to 1.7 mls with ddH2O
add 2 g CsCl, 80 ul EtBr (10 mg/ml)
load TL 100 tubes, cent. 100 krpm x >6 hrs, 20 C

NOTES:

TS is:	50 mM Tris-Cl (8.0)
	50 mM NaCl

TES is: TS plus 10 mM EDTA

sucrose mix:	1.6 M sucrose
		0.55 M Tris-Cl (8.0)
		0.10 M EDTA