From a 10-cm plates of cells that are almost confluent
á Withdraw media
¥ Replace with 4 mL 1X SET (1% SDS, 10 mM Tris, 5 mM EDTA, pH 7.5), 150 mM NaCL, plus 50-100 ug/mL proteinase K.
¥ Incubate an hour or more at 37C (in incubator)
¥ Pipet solution upp and down with 10 mL pipet - squirt out vigoruosly until solution flows uniformly (It should be viscous as snot at first - pipeting 5-6 times usually enough; if it is difficult to pipet, repeat more times)
¥ Transfer to 14 mL polypropylene tube (e.g. Falcon 2059)
¥ Add 3-4 mL 50:50 mixture phenol and chloroform
¥ Shake vigoursly
¥ Cfg to separate phases (table top cfg usually enough)
¥ Withdraw upper aqueous phase with wide bore pipet (I use the wrong end of a 2 mL disposible pipet) Avoid interface if there is anything visible. Don't worry about getting last bit of aqueous phase.
¥ Transfer to to new 14 mL tube
¥ Add 2 vol Ethanol
¥ Mix well (you should see stringy ppt form)
¥ Cfg (5 min 9K rpm) (I use Sorvall swinging bucket rotor HB6)
¥ Decant supernatant (make sure pellet doesn't slip out)
¥ Rinse with 70% Ethanol, 50 mM NaCl
¥ Drain for 10 min
¥ Resuspend DNA in 400 uL TE (vortex and let sit ~ 30 min)
¥ Transfer with 1 mL pipet tip to 1.5 mL Eppi tube. (should be a little viscous but pipet smoothly)
¥ Measure DNA concentration (should be about 500 ug/mL)
¥ Digest 10 uL (5 ug) in 25 uL reaction.