(2/18/06) The following plasmid prep is from Zhiqiang Zhang it produces DNA that can be relibly sequenced by our facility.  On of the key steps is the PEG precipitation.  This technique was pioneered by John Lis and his original paper notes that the DNA concentration has to be above 100 ug/ml to precipitate efficiently.  Keep this in mind if you are working with a low copy number plasmid.  I came across exactly the same procedure at several locations on the Web: site 1, site 2.  The first procedure recommends against autoclaving your PEG.  My recommendation is to not autoclave it and store it in the refrigerator.


Grow cells
1. Innoculate 2ml of LB+antibiotic with cells from a single colony and grow overnight at 37^oC.
2. Transfer 0.5 ml of the overnight culture to 20 ml of LB+antibiotic, and grow 4 to 8 hrs at 37^oC.
3. Harvest cells in the centrifuge.

Alkaline lysis
1. Resuspend the bacterial pellet in 1 ml of Solution I (50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA).
2. Add 1 ml of Solution II (0.2 M NaOH, 1% SDS), mix it by inverting the tube rapidly 5 times.  Store the tube for 5 min at room temperature.
3. Add 1.5 ml Solution III (made by combining 60 ml of 5M potassium acetate, 11.5 ml glacial acetic acid, 28.5ml H₂O) and mix gently by inverting the tube.  Store the tube on ice for 5 min.
4. Centrifuge at 12,000 x g for 5 min, transfer the supernatant to a fresh tube.
5. Extract it with an equal volume of phenol:chloroform (1:1).
6. Extract it with equal volume of chloroform:isopropanol (24:1).
7. Preciptate DNA with 2.5 volume of ethanol at room temperature.
8. Wash the pellet with 70% ethanol.

Precipitate by PEG8000
1. Dissolve DNA pellet in 80 ul H₂O, and precipitate the plasmid DNA by first adding 20 ul of 4M NaCl, and then adding 100 ul of 13% PEG 8000.
2. After thorough mixing, incubate the sample on ice for 20 min, then centrifuge for 15 min at 4^oC.
3. Remove the supernatant, wash the pellet with 500 ul of 70% ethanol.
4. Dry the pellet, and resuspend in suitable volume of H₂O.