From a 10-cm plates of cells that are almost confluent


    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.