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1. Resuspend pellets in 100 ul of Solution I (Tris-EDTA-glucose, 50mM glucose, 10mM EDTA,
25mM Tris pH 8.0 - storage at 4?C) place on ice for 5 minutes. Add 150ul of ice-cold Solution III (for 100ml, 60ml of 5M KOAc, 11.5ml glacial Acetic Acid,
28.5ml H2O) and mix by inverting the tube gently.Grow overnight cultures of E. coli (1.5-2.0 ml) in LB broth at 37?C using appropriate antibiotic
selection.Harvest cells in microfuge tube by centrifuging for 5 minutes at 4,000 rpm, Discard media.Dry pellets (5 minutes in speedvac) and resuspend in 20-40ul TE + RNase A. Shaking for ~ 10
minutes was done to resuspend DNA pellets.Add 200 ul of freshly prepared Solution II (0.2N NaOH, 1%SDS).The solution mixture should be turbid, incubate
on ice for 5 min.Chloroform will
remove soluble proteins from the preps.2.3.4.5.6.7.8.9.10.11.12.13.


Original text


  1. Grow overnight cultures of E. coli (1.5-2.0 ml) in LB broth at 37˚C using appropriate antibiotic
    selection.

  2. Harvest cells in microfuge tube by centrifuging for 5 minutes at 4,000 rpm, Discard media.

  3. Resuspend pellets in 100 µl of Solution I (Tris-EDTA-glucose, 50mM glucose, 10mM EDTA,
    25mM Tris pH 8.0 – storage at 4˚C) place on ice for 5 minutes.

  4. Add 200 µl of freshly prepared Solution II (0.2N NaOH, 1%SDS). Mix by inversion three times
    without vortex. Place on ice for 5 minutes, not more than that.

  5. Add 150µl of ice-cold Solution III (for 100ml, 60ml of 5M KOAc, 11.5ml glacial Acetic Acid,
    28.5ml H2O) and mix by inverting the tube gently. The solution mixture should be turbid, incubate
    on ice for 5 min.

  6. Spin for 5 minutes at 12,000 rpm to remove precipitate. Transfer supernatant to a clean tube (~
    450µl).

  7. Add equal volume of chloroform to the supernatant. Mix on vortex for 1 minute. Chloroform will
    remove soluble proteins from the preps.

  8. Spin for 5 minutes at 10,000 rpm. Transfer the top aqueous phase to a clean tube.

  9. Add 1.0ml of 100% EtOH to the supernatants and place in -20˚C for at least 20 minutes or
    overnight.

  10. Spin samples for 15 minutes at 14,000 rpm and discard supernatant carefully not to disturb DNA
    pellet.

  11. Wash pellets with 0.5-1.0ml of 70% EtOH, spin for 10 minutes at 14,000 rpm.

  12. Dry pellets (5 minutes in speedvac) and resuspend in 20-40µl TE + RNase A. Shaking for ~ 10
    minutes was done to resuspend DNA pellets. DNA is incubated at room temperature for 4 hours or
    at 37˚C for 1 hour to degrade RNA before freezing. Typical mini-preps should yield around 5-
    20µg.

  13. DNA is stored at -20˚C.


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